Methods for treating hepatitis c

ABSTRACT

In accordance with the present invention, compounds that can inhibit viral replication, preferably Hepatitis C Virus (HCV) replication, have been identified, and methods for their use provided. In one aspect of the invention, compounds useful in the treatment or prevention of a viral infection are provided. In another aspect of the invention, compounds useful in the treatment or prevention of HCV infection are provided.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/589,876, filed Jul. 22, 2004, which application is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods for treating Hepatitis C usingthienopyridine compounds that modify translational control of HepatitisC virus.

BACKGROUND OF THE INVENTION

An estimated 170 million people worldwide are reported to be infectedwith hepatitis C virus (HCV), the causative agent of hepatitis C.Seventy to eighty percent of HCV infections lead to chronic liverinfection, which in turn may result in severe liver disease, includingliver fibrosis, cirrhosis, and hepatocellular carcinoma (115).

HCV constitutes the Hepacivirus genus of the family Flaviviridae (106),and contains a positive-stranded 9.6 kb RNA genome. The features of theHCV genome include a 5′-untranslated region (UTR) that encodes aninternal ribosome entry site (IRES) that directs the translation of asingle open reading frame (ORF) encoding a polyprotein of 3,010 aminoacids. The HCV ORF is followed by a 3′-UTR of variable length, dependingon the HCV variant, that encodes the sequences required for theinitiation of antigenomic strand synthesis (79).

The HCV IRES and 3′-UTR both contain regions of RNA structures that arerequired for genome translation and replication. The HCV polyprotein isposttranslationally processed into at least 10 mature viral proteins,including the structural proteins core (putative nucleocapsid), E1 andE2 and the nonstructural (NS) proteins NS2 to NS5B.

Three distinct elements have been shown to be involved in HCVIRES-mediated translation: (1) integrity of the global structure of HCVIRES, (2) the 3′-terminal region of the HCV genome; and (3) trans-actingcellular factors that interact with the HCV IRES element and assist intranslation initiation (35).

The initiation of protein synthesis in eukaryotic cells predominantlyfollows the 5′ cap-dependent, first AUG rule (61). However, anincreasing number of viral (6, 12, 28, 31a, 50, 95, 97, 98, 105, 128)and cellular mRNAs (18, 39, 45, 78, 91, 130) have been shown to use anIRES element to direct translation initiation. In 1992, an IRES elementwas reported in the 5′ UTR of the HCV RNA genome (129), indicating thatsynthesis of the viral protein is initiated in a cap-independentfashion.

A bicistronic expression system can be used to define and evaluate thefunction of IRES elements. This test system harbors two differentreporter genes in which the 5′-proximal reporter gene is expressed by acap dependent translation mechanism while the second reporter isexpressed only if an upstream sequence inserted in the intergenic spacecontains an IRES sequence element. Using this system, a putative IRES inthe HCV 5′ UTR was unambiguously demonstrated to function as an IRESinvolved in translational control of viral proteins (133). In vitrotranslation, RNA transfection, and mutagenesis studies provided furtherevidence that the HCV 5′ UTR contains an IRES element (23, 41, 42, 108,129, 132, 133, 134). Both in vitro and cell-based studies demonstratedthat the HCV IRES guides cellular translation initiation factors to aninternal site of the viral RNA (56, 58, 120), thus functionallydemonstrating the HCV IRES activity. Taken together, these resultsdemonstrate that the HCV 5′-UTR contains an IRES element that plays anactive and crucial role in the mechanism of internal initiation for HCVprotein translation.

The IRES is one of the most conserved regions of the HCV genome,reflecting its essential nature for viral replication and proteinsynthesis (13, 118, 122). Although both 5′ and 3′ sequences of the IRESappear to play a role in the control of initiation of translation (42,109, 110, 113, 136), the minimal sequence requirement for HCV IRESfunction has been mapped to a region between nucleotides 44-354 (40).

Biochemical probing and computer modeling indicate that the HCV IRES andits 5′ sequence are folded into a distinct structure that consists offour major domains and a pseudoknot (11, 42, 122). Domain I contains asmall stem-loop structure that does not appear to be a functional partof the IRES element while domains II, III, and IV contain the HCV TRESactivity (43, 111). The relationships between secondary and tertiarystructures of the HCV IRES and their function have recently beenestablished (5, 55, 56, 99, 124). Both domains II and III consist ofmultiple stems, loops, and bulges and are important for IRES activity(23, 40, 51, 52, 54, 56, 64, 74, 75, 93, 107, 108, 110, 124, 127, 131,139). Domain II can induce conformational changes on the ribosome thathave been implicated in the decoding process (124). Domain III has thehighest degree of structural conservation among the different HCVstrains. It comprises the core of the flavivirus IRES and has 6subdomains (40). Various studies have shown that subdomain IIId formscomplex secondary/tertiary structures and is critical for initiationactivity (55, 56, 57, 124, 129). Domain IV has one stem-loop that spansthe initiation codon and is specific for the HCV IRES (41, 122), but theprecise role of domain IV in IRES activity remains controversial (41,112).

The role of the HCV IRES is to position the translational machinery nearan internal initiator codon in the viral mRNA. The translationinitiation mechanism of the HCV IRES differs significantly from that of5′-cap-dependent translation initiation (7, 21, 31, 35, 81, 96, 114,123). Most cellular capped mRNAs utilize a number of initiation factors(eIFs) that are required for the translation initiation process. Theinitial steps of the process require proteins that interact with the 5′cap structure and recruit the 40S ribosomal subunit to the cap-proximalregion of mRNA. This complex then scans 3′ of the cap, until reaching anAUG codon at which translation will initiate (21, 114). However, in thecase of HCV, the IRES functionally replaces the 5′ cap structure,allowing the 40S ribosomal subunit and eIF3 to bind directly to the RNA.Subdomain IIId of the HCV IRES harbors the binding site for the 40Sribosomal subunit and the only initiation factors required fortranslation initiation are eIF2, eIF3, and eIF4E (15, 58, 94, 100, 120,124).

The polypyrimidine track-binding protein (PTB) and La autoantigen arenoncanonical translation initiation factors that bind to HCV IRES andenhance its activity (1, 2, 3, 4, 5, 30, 48, 49, 53). PTB, a 57-kDaprotein involved in RNA splicing, is also necessary for efficientIRES-mediated translation initiation of picornavirus mRNA, and somecellular mRNAs (10, 11, 36, 53, 59, 89, 92). The La autoantigen, a 52kDa double-stranded RNA unwinding protein, also increases the activityof poliovirus and cellular IRESs (38, 85, 86). Other cellular factorsinvolved in HCV IRES-mediated translation initiation include proteasomeα-subunit PSMA7 (62), ribosomal protein S5 (26), ribosomal protein S9(24, 25, 100), and hnRNPL (33). However, the role of these RNA-bindingproteins in HCV IRES-mediated initiation of translation is unclear.Recently, it was reported that the activity of interferon (IFN) αagainst HCV replication might target HCV IRES-mediated translationinitiation by causing a reduction of La protein levels (117). Thus, aninhibitor that blocks interaction between the IRES and the noncanonicalfactors might efficiently inhibit HCV replication and lack cytotoxicity.

Currently, only interferon (IFN) α and the nucleoside analogueribavirin, in combination, are marketed for the treatment of HCVinfection. However, these two agents are immunomodulators and havelimited efficacy, relatively high toxicity, and high cost (80, 83, 84,138). Although the treatment outcome is variable among the six major HCVgenotypes, only about one-half of all treated patients respond totherapy, suggesting that the virus encodes protein products that maydirectly or indirectly attenuate the antiviral action of IFN. IFNs arenaturally produced in response to virus infection, and cellular exposureto IFN leads to the induced expression of a variety of IFN-stimulatedgenes (ISGs), many of which have an antiviral function. ISG action canlimit virus replication at multiple points within the replicative cycle.

There remains a need for a more effective means of treating patientsafflicted with HCV. Specifically, a need exists for novel antiviraldrugs that do not elicit cross-resistance to existing treatmentmodalities, and which demonstrate synergy with other anti-HCV agents.The applicants set out to identify drug candidates that inhibit HCVinfection and were successful in identifying Indole compounds that areuseful as anti-HCV agents. Without being limited to one theory, it isbelieved that the compounds of the present invention inhibitIRES-mediated initiation, elongation, and termination, i.e. translation.

The compounds of the present invention may also be useful for inhibitingtranslation of other cap-independent viruses that contain an IRESelement. Such viruses include those of the picornavirus genus, such aspoliovirus, hepatitis A virus and rhinovirus; those of the coronavirusgenus, such as SARS; those of the arbovirus genus; those of theflavivirus genus, such as yellow fever, dengue, and West Nile virus,herpesviruses, such as herpes simplex virus and Kaposi'ssarcoma-associated herpesvirus, or any other virus with a similar modeof replication. Furthermore, compounds of the invention may also beuseful for inhibiting HIV, or any other virus with a similar mode oftranslation.

All documents referred to herein are incorporated by reference into thepresent application as though fully set forth herein.

SUMMARY OF THE INVENTION

In accordance with the present invention, compounds that inhibit HCVinfection have been identified, and methods for their use provided.

In one aspect of the invention, compounds of Formula (I) are providedwhich are useful in the prevention and/or treatment of HCV infection.Without being limited to one theory, it is believed that the compoundsof the present invention inhibit IRES-mediated initation, elongation andtermination, i.e., translation. The compounds of Formula (I) may also beuseful for inhibiting and/or treating other viral infections where thevirus contains an IRES element. Such viruses include those of thepicornavirus genus, such as poliovirus, hepatitis A virus andrhinovirus; those of the coronaviridae genus, such as SARS; those of thearbovirus genus; those of the flavivirus genus, such as yellow fever,dengue, and West Nile virus, herpesviruses, such as herpes simplex virusand Kaposi's sarcoma-associated herpesvirus, or any other virus with asimilar mode of replication. Furthermore, compounds of the invention mayalso be useful for inhibiting HIV, or any other virus with a similarmode of translation.

In another aspect of the invention, methods are provided for theprevention and/or treatment of HCV infection.

In yet another aspect of the invention, pharmaceutical compositionscomprising the compounds of the invention for the prevention and/ortreatment of HCV infection are provided.

In one embodiment, the invention is directed to methods for inhibitingHCV IRES-mediated initiation and translation comprising administering anamount of at least one compound of the invention, effective forinhibiting IRES-mediated initiation and translation, to a subject inneed thereof.

Exemplary Embodiments

-   Embodiment 1. A pharmaceutical composition for the prevention and/or    treatment of Hepatitis C viral (HCV) infection comprising a    therapeutically effective amount of at least one compound having the    following formula:

wherein:

-   X is:-   hydrogen;-   a cyano group;-   an amino group;-   a

or

-   a 5- or 6-membered heteroaryl;-   a C₆ to C₈ aryl, optionally substituted with:

an alkoxy group,

a cyano group, or

a halogen;

-   or X together with Y forms:

-   Y is:-   a halogen;-   an amino group;-   a

or

-   a —SO₂R_(x), where R_(x) is a C₁ to C₆ alkyl;-   a cyano group;-   a —COOR_(x) group, where R_(x) is a C₁ to C₆ alkyl;-   a C₆ to C₈ aryl, optionally substituted with:

an alkoxy; or

a cyano group;

-   a —COR_(a) group, where R_(a) is:

an amino optionally substituted with one or two C₁ to C₆ alkyls, wherethe alkyls are optionally substituted with a C₆ to C₈ aryl;

a —NHR_(b) group where R_(b) is:

-   -   a C₆ to C₈ aryl optionally substituted with:        -   a haloalkyl; or        -   a halogen        -   a haloalkoxy; or

a 5- or 6-membered heterocycle optionally substituted with a C₁ to C₆alkyl;

a C₁ to C₆ alkyl;

a —SR_(x) group, where R_(x) is as defined above;

-   a 5 or 6 membered heteroaryl optionally substituted with:

a C₆ to C₈ aryl optionally substituted with:

-   -   an alkoxy    -   a halogen; or    -   a C₁ to C₆ alkyl;

a 5- or 6-membered heteroaryl optionally substituted with

-   -   an alkoxy    -   a halogen; or    -   a C₁ to C₆ alkyl;

-   a C₁ to C₆ alkyl, optionally substituted with a —OR_(c), where R_(c)    is a C₆ to C₈ aryl optionally substituted with one or more halogens;    or

-   a nitro group;

-   a —NHR_(d) group, where R_(d) is a C₆ to C₈ aryl optionally    substituted with an alkoxy

-   a —NHCOR_(e) group where R_(e) is:

a C₆ to C₈ aryl optionally substituted with a haloalkyl;

a C₁ to C₆ alkyl;

-   or together with X forms:

-   R is:-   a hydrogen-   a haloalkyl;-   a C₁ to C₆ alkyl optionally substituted with hydroxyl;-   a 5- or 6-member heteroaryl;-   a C₆ to C₈ aryl optionally substituted with one or more halogens;-   or R together with R₁ forms:

-   R₁ is:-   a hydrogen;-   a C₆ to C₈ aryl-   a C₁ to C₆ alkyl;-   a OCOR_(f) where R_(f) is a 5- or 6-membered heterocycle;-   an alkoxy optionally substituted with an amino group, wherein the    amino group is optionally substituted with one or two C₁ to C₆    alkyls, where the alkyls are optionally substituted with an amino    optionally substituted with one or two C₁ to C₆ alkyls-   an alkoxy optionally substituted with a 5 to 8 membered heterocycle    optionally substituted with a C₁ to C₆ alkyl, which is optionally    substituted with:

an alkoxy, or

an amino, optionally substituted with one or two C₁ to C₆ alkyls;

-   or R₁ together with R₂ forms:

-   R₁ together with R forms:

-   R₂ is:-   a C₁ to C₆ alkyl;-   a 5 or 6-membered heterocycle;-   an amino optionally substituted with a C₁ to C₆ alkyl;-   or R₁ together with R₂ forms:

or a pharmaceutically acceptable salt thereof, together with apharmaceutically acceptable excipient and optionally an additionalanti-HCV agent.

-   Embodiment 2. The pharmaceutical composition of Embodiment 1,    wherein said additional anti-HCV agent is selected from the group    consisting of pegylated interferon, un-pegylated interferon,    ribavirin or prodrugs or derivatives thereof, a glucosidase    inhibitor, a protease inhibitor, a polymerase inhibitor, p7    inhibitors, an entry inhibitor, a fusion inhibitor, an    anti-fibrotic, a caspase inhibitor, a drug which targets inosine    monophosphate dehydrogenase inhibitors (IMPDH), synthetic thymosin    alpha 1, therapeutic vaccines, immunomodulators, a helicase    inhibitor, and a Toll-like receptor agonist.-   Embodiment 3. The pharmaceutical composition of Embodiment 1,    wherein X is an amino group or a hydrogen.-   Embodiment 4. The pharmaceutical composition of Embodiment 1,    wherein Y is a 5 or 6 membered heteroaryl optionally substituted    with:

a C₆ to C₈ aryl optionally substituted with:

-   -   an alkoxy    -   a halogen; or    -   a C₁ to C₆ alkyl; or

a 5- or 6-membered heteroaryl optionally substituted with

-   -   a halogen.

-   Embodiment 5. The pharmaceutical composition of Embodiment 1,    wherein Y is a —COOR_(x) group, where R_(x) is as defined above;

-   a —COR_(a) group, where R_(a) is:

an amino optionally substituted with one or two C₁ to C₆ alkyls, wherethe alkyls are optionally substituted with a C₆ to C₈ aryl;

a —SR_(x) group, where R_(x) is as defined above;

-   a 5 or 6 membered heteroaryl optionally substituted with:

a C₆ to C₈ aryl optionally substituted with:

-   -   an alkoxy    -   a halogen; or    -   a C₁ to C₆ alkyl;    -   a 5- or 6-membered heteroaryl optionally substituted with a C₆        to C₈ aryl optionally substituted with a halogen;

-   Embodiment 6. The pharmaceutical composition of Embodiment 1,    wherein R is a C₁ to C₆ alkyl.

-   Embodiment 7. The pharmaceutical composition of Embodiment 6,    wherein R is a methyl group.

-   Embodiment 8. The pharmaceutical composition of Embodiment 6,    wherein R, R₁ and R₂ are independently C₁ to C₆ alkyl.

-   Embodiment 9. The pharmaceutical composition of Embodiment 6,    wherein said C₁ to C₆ alkyl in R, R₁ and R₂ is independently a    methyl or an ethyl.

-   Embodiment 10. The pharmaceutical composition of Embodiment 1,    wherein R₁ is selected from the group consisting of

-   a C₁ to C₆ alkyl; and

-   an alkoxy optionally substituted with an amino group, wherein the    amino group is optionally substituted with one or two C₁ to C₆    alkyls, where the alkyls are optionally substituted with an amino    optionally substituted with one or two C₁ to C₆ alkyls; and

-   an alkoxy optionally substituted with a 5 to 8 membered heterocycle    optionally substituted with a C₁ to C₆ alkyl, which is optionally    substituted with:

an amino, optionally substituted with one or two C₁ to C₆ alkyls.

-   Embodiment 11. The pharmaceutical composition of Embodiment 10,    wherein R₁ is—a C₁ to C₆ alkyl.-   Embodiment 12. The pharmaceutical composition of Embodiment 11,    wherein R₁ is methyl or ethyl.-   Embodiment 13. The pharmaceutical composition of Embodiment 12,    wherein R₁ is methyl or ethyl.-   Embodiment 14. The pharmaceutical composition of Embodiment 1,    wherein R₂ is —a C₁ to C₆ alkyl.-   Embodiment 15. The pharmaceutical composition of Embodiment 14,    wherein R₂ is methyl.

Embodiment 16. The pharmaceutical composition of Embodiment 1, whereinsaid compound is selected from the group consisting of the followingcompounds:

-   Embodiment 17. The pharmaceutical composition of Embodiment 1,    wherein said compound is selected from the group consisting of the    following compounds:

-   Embodiment 18. A pharmaceutical composition for the prevention or    treatment of

Hepatitis C viral (HCV) infection comprising at least one of thefollowing compounds:

or a pharmaceutically acceptable salt thereof, together with apharmaceutically acceptable excipient.

-   Embodiment 19. A pharmaceutical composition for the prevention or    treatment of Hepatitis C viral (HCV) infection comprising at least    one of the following compounds:

or a pharmaceutically acceptable salt thereof, together with apharmaceutically acceptable excipient.

-   Embodiment 20. The pharmaceutical composition of Embodiment 17,    wherein said composition further comprises an additional anti-HCV    agent selected from the group consisting of pegylated interferon,    un-pegylated interferon, ribavirin or prodrugs or derivatives    thereof, a glucosidase inhibitor, a protease inhibitor, a polymerase    inhibitor, p7 inhibitors, an entry inhibitor, a fusion inhibitor, an    anti-fibrotic, a caspase inhibitor, a drug which targets inosine    monophosphate dehydrogenase inhibitors (IMPDH), synthetic thymosin    alpha 1, therapeutic vaccines, immunomodulators, a helicase    inhibitor, and a Toll-like receptor agonist.-   Embodiment 21. A pharmaceutical composition for the prevention or    treatment of Hepatitis C viral (HCV) infection comprising a compound    having the following formula:

wherein

-   X is amino or hydrogen;-   Y is-   a —COOR_(x) group, where R_(x) is as defined above;-   a —COR_(a) group, where R_(a) is:

an amino optionally substituted with one or two C₁ to C₆ alkyls, wherethe alkyls are optionally substituted with a C₆ to C₈ aryl;

a —SR_(x) group, where R_(x) is as defined above;

-   a 5 or 6 membered heteroaryl optionally substituted with:

a C₆ to C₈ aryl optionally substituted with:

-   -   an alkoxy    -   a halogen; or    -   a C₁ to C₆ alkyl;    -   a 5- or 6-membered heteroaryl optionally substituted with a C₆        to C₈ aryl optionally substituted with a halogen;

-   R is a C₁ to C₆ alkyl;

-   R₁ is a C₁ to C₆ alkyl or R₁ is selected from the group consisting    of

-   an alkoxy optionally substituted with an amino group, wherein the    amino group is optionally substituted with one or two C₁ to C₆    alkyls, where the alkyls are optionally substituted with an amino    optionally substituted with one or two C₁ to C₆ alkyls; and

-   an alkoxy optionally substituted with a 5 to 8 membered heterocycle    optionally substituted with a C₁ to C₆ alkyl, which is optionally    substituted with:

an amino, optionally substituted with one or two C₁ to C₆ alkyls; and

-   R₂ is —a C₁ to C₆ alkyl or an amino optionally substituted with a C₁    to C₆ alkyl;    or a pharmaceutically acceptable salt thereof, together with an    additional anti-HCV agent and a pharmaceutically acceptable    excipient.-   Embodiment 22. The pharmaceutical composition of Embodiment 21,    wherein said additional anti-HCV agent is selected from the group    consisting of pegylated interferon, un-pegylated interferon,    ribavirin or prodrugs or derivatives thereof, a glucosidase    inhibitor, a protease inhibitor, a polymerase inhibitor, p7    inhibitors, an entry inhibitor, a fusion inhibitor, an    anti-fibrotic, a caspase inhibitor, a drug which targets inosine    monophosphate dehydrogenase inhibitors (IMPDH), synthetic thymosin    alpha 1, therapeutic vaccines, immunomodulators, a helicase    inhibitor, and a Toll-like receptor agonist.-   Embodiment 23. The pharmaceutical composition of Embodiment 21,    wherein R is a methyl group.-   Embodiment 24. The pharmaceutical composition of Embodiment 21,    wherein R, R₁ and R₂ are independently C₁ to C₆ alkyl.-   Embodiment 25. The pharmaceutical composition of Embodiment 24,    wherein said C₁ to C₆ alkyl in R, R₁ and R₂ is independently a    methyl or an ethyl.-   Embodiment 26. The pharmaceutical composition of Embodiment 21,    wherein R₁ is a C₁ to C₆ alkyl.-   Embodiment 27. The pharmaceutical composition of Embodiment 26,    wherein R₁ is methyl or ethyl.-   Embodiment 28. The pharmaceutical composition of Embodiment 21,    wherein R₂ is a C₁ to C₆ alkyl.-   Embodiment 29. The pharmaceutical composition of Embodiment 21,    wherein R₂ is methyl.-   Embodiment 30. A method for treating a subject for a Hepatitis C    viral (HCV) infection, or for preventing a subject from becoming    infected with HCV, comprising administering to said subject a    pharmaceutical composition comprising an HCV inhibitory amount of at    least one compound having the following formula:

wherein:

-   X is:-   hydrogen;-   a cyano group;-   an amino group;-   a

or

-   a 5- or 6-membered heteroaryl;-   a C₆ to C₈ aryl, optionally substituted with:

an alkoxy group,

a cyano group, or

a halogen;

-   or X together with Y forms:

-   Y is:-   a halogen;-   an amino group;-   a

or

-   a —SO₂R_(x), where R_(x) is a C₁ to C₆ alkyl;-   a cyano group;-   a —COOR_(x) group, where R_(x) is a C₁ to C₆ alkyl;-   a C₆ to C₈ aryl, optionally substituted with:

an alkoxy; or

a cyano group;

-   a —COR_(a) group, where R_(a) is:

an amino optionally substituted with one or two C₁ to C₆ alkyls, wherethe alkyls are optionally substituted with a C₆ to C₈ aryl;

a —NHR_(b) group where R_(b) is:

-   -   a C₆ to C₈ aryl optionally substituted with:        -   a haloalkyl; or        -   a halogen        -   a haloalkoxy; or

a 5- or 6-membered heterocycle optionally substituted with a C₁ to C₆alkyl;

a C₁ to C₆ alkyl;

a —SR_(x) group, where R_(x) is as defined above;

-   a 5 or 6 membered heteroaryl optionally substituted with:

a C₆ to C₈ aryl optionally substituted with:

-   -   an alkoxy    -   a halogen; or    -   a C₁ to C₆ alkyl;

a 5- or 6-membered heteroaryl optionally substituted with

-   -   an alkoxy    -   a halogen; or    -   a C₁ to C₆ alkyl;

-   a C₁ to C₆ alkyl, optionally substituted with a —OR_(c), where R_(c)    is a C₆ to C₈ aryl optionally substituted with one or more halogens;    or

-   a nitro group;

-   a —NHR_(d) group, where R_(d) is a C₆ to C₈ aryl optionally    substituted with an alkoxy

-   a —NHCOR_(e) group where R_(e) is:

a C₆ to C₈ aryl optionally substituted with a haloalkyl;

a C₁ to C₆ alkyl;

-   or together with X forms:

-   R is:-   a hydrogen-   a haloalkyl;-   a C₁ to C₆ alkyl optionally substituted with hydroxyl;-   a 5- or 6-member heteroaryl;-   a C₆ to C₈ aryl optionally substituted with one or more halogens;-   or R together with R₁ forms:

-   R₁ is:-   a hydrogen;-   a C₆ to C₈ aryl-   a C₁ to C₆ alkyl;-   a OCOR_(f) where R_(f) is a 5- or 6-membered heterocycle;-   an alkoxy optionally substituted with an amino group, wherein the    amino group is optionally substituted with one or two C₁ to C₆    alkyls, where the alkyls are optionally substituted with an amino    optionally substituted with one or two C₁ to C₆ alkyls-   an alkoxy optionally substituted with a 5 to 8 membered heterocycle    optionally substituted with a C₁ to C₆ alkyl, which is optionally    substituted with:

an alkoxy, or

an amino, optionally substituted with one or two C₁ to C₆ alkyls;

-   or R₁ together with R₂ forms:

-   R₁ together with R forms:

-   R₂ is:-   a C₁ to C₆ alkyl;-   a 5 or 6-membered heterocycle;-   an amino optionally substituted with a C₁ to C₆ alkyl;-   or R₁ together with R₂ forms:

or a pharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable excipient.

-   Embodiment 31. The method of Embodiment 30, wherein said method    further comprises administering an additional anti-HCV agent.-   Embodiment 32. The method of Embodiment 30, wherein said additional    anti-HCV agent is selected from the group consisting of pegylated    interferon, un-pegylated interferon, ribavirin or prodrugs or    derivatives thereof, a glucosidase inhibitor, a protease inhibitor,    a polymerase inhibitor, p7 inhibitors, an entry inhibitor, a fusion    inhibitor, an anti-fibrotic, a caspase inhibitor, a drug which    targets inosine monophosphate dehydrogenase inhibitors (IMPDH),    synthetic thymosin alpha 1, therapeutic vaccines, immunomodulators,    a helicase inhibitor, and a Toll-like receptor agonist.-   Embodiment 33. The method of Embodiment 30, wherein X is an amino    group or a hydrogen.-   Embodiment 34. The method of Embodiment 30, wherein Y is a 5 or 6    membered heteroaryl optionally substituted with:

a C₆ to C₈ aryl optionally substituted with:

-   -   an alkoxy    -   a halogen; or    -   a C₁ to C₆ alkyl; or

a 5- or 6-membered heteroaryl optionally substituted with

-   -   a halogen.

-   Embodiment 35. The method of Embodiment 30, wherein Y is a —COOR_(x)    group, where R_(x) is as defined above;

-   a —COR_(a) group, where R_(a) is:

an amino optionally substituted with one or two C₁ to C₆ alkyls, wherethe alkyls are optionally substituted with a C₆ to C₈ aryl;

a —SR_(x) group, where R_(x) is as defined above;

-   a 5 or 6 membered heteroaryl optionally substituted with:

a C₆ to C₈ aryl optionally substituted with:

-   -   an alkoxy    -   a halogen; or    -   a C₁ to C₆ alkyl;    -   a 5- or 6-membered heteroaryl optionally substituted with a C₆        to C₈ aryl optionally substituted with a halogen;

-   Embodiment 36. The method of Embodiment 30, wherein R is a C₁ to C₆    alkyl.

-   Embodiment 37. The method of Embodiment 36, wherein R is a methyl    group.

-   Embodiment 38. The method of Embodiment 30, wherein R, R₁ and R₂ are    independently C₁ to C₆ alkyl.

-   Embodiment 39. The method of Embodiment 38, wherein said C₁ to C₆    alkyl in R, R₁ and R₂ is independently a methyl or an ethyl.

-   Embodiment 40. The method of Embodiment 30, wherein R₁ is selected    from the group consisting of

-   a C₁ to C₆ alkyl; and

-   an alkoxy optionally substituted with an amino group, wherein the    amino group is optionally substituted with one or two C₁ to C₆    alkyls, where the alkyls are optionally substituted with an amino    optionally substituted with one or two C₁ to C₆ alkyls; and

-   an alkoxy optionally substituted with a 5 to 8 membered heterocycle    optionally substituted with a C₁ to C₆ alkyl, which is optionally    substituted with:

an amino, optionally substituted with one or two C₁ to C₆ alkyls.

-   Embodiment 41. The method of Embodiment 40, wherein R₁ is—a C₁ to C₆    alkyl.-   Embodiment 42. The method of Embodiment 41, wherein R₁ is methyl or    ethyl.-   Embodiment 43. The method of Embodiment 30, wherein R₂ is—a C₁ to C₆    alkyl.-   Embodiment 44. The method of Embodiment 43, wherein R₂ is methyl.-   Embodiment 45. The method of Embodiment 30, wherein said compound is    selected from the group consisting of the following compounds:

-   Embodiment 46. The method of Embodiment 30, wherein said compound is    selected from the group consisting of the following compounds:

-   Embodiment 47. A pharmaceutical composition for the prevention or    treatment of Hepatitis C viral (HCV) infection comprising at least    one of the following compounds:

or a pharmaceutically acceptable salt thereof, together with apharmaceutically acceptable excipient.

-   Embodiment 48. A method for the treatment of for the prevention or    treatment of Hepatitis C viral (HCV) infection comprising at least    one of the following compounds:

-   Embodiment 49. A method for treating a subject for a Hepatitis C    viral (HCV) infection, or for preventing a subject from being    infected with HCV, comprising administering to said subject a    pharmaceutical composition comprising an HCV inhibitory amount of a    compound having the following formula:

wherein

-   X is amino or hydrogen;-   Y is-   a —COOR_(x) group, where R_(x) is as defined above;-   a —COR_(a) group, where R_(a) is:

an amino optionally substituted with one or two C₁ to C₆ alkyls, wherethe alkyls are optionally substituted with a C₆ to C₈ aryl;

a —SR_(x) group, where R_(x) is as defined above;

-   a 5 or 6 membered heteroaryl optionally substituted with:

a C₆ to C₈ aryl optionally substituted with:

-   -   an alkoxy    -   a halogen; or    -   a C₁ to C₆ alkyl;    -   a 5- or 6-membered heteroaryl optionally substituted with a C₆        to C₈ aryl optionally substituted with a halogen;

-   R is a C₁ to C₆ alkyl;

-   R₁ is a C₁ to C₆ alkyl or R₁ is selected from the group consisting    of

-   an alkoxy optionally substituted with an amino group, wherein the    amino group is optionally substituted with one or two C₁ to C₆    alkyls, where the alkyls are optionally substituted with an amino    optionally substituted with one or two C₁ to C₆ alkyls; and

-   an alkoxy optionally substituted with a 5 to 8 membered heterocycle    optionally substituted with a C₁ to C₆ alkyl, which is optionally    substituted with:

an amino, optionally substituted with one or two C₁ to C₆ alkyls; and

-   R₂ is—a C₁ to C₆ alkyl or an amino optionally substituted with a C₁    to C₆ alkyl;    or a pharmaceutically acceptable salt thereof and a pharmaceutically    acceptable excipient.-   Embodiment 50. The method of Embodiment 49, wherein said method    further comprises administering an additional anti-HCV agent.-   Embodiment 51. The method of Embodiment 50, wherein said additional    anti-HCV agent is selected from the group consisting of pegylated    interferon, un-pegylated interferon, ribavirin or prodrugs or    derivatives thereof, a glucosidase inhibitor, a protease inhibitor,    a polymerase inhibitor, p7 inhibitors, an entry inhibitor, a fusion    inhibitor, an anti-fibrotic, a caspase inhibitor, a drug which    targets inosine monophosphate dehydrogenase inhibitors (IMPDH),    synthetic thymosin alpha 1, therapeutic vaccines, immunomodulators,    a helicase inhibitor, and a Toll-like receptor agonist.-   Embodiment 52. A method for treating a subject for a Hepatitis C    viral (HCV) infection comprising administering to said subject a    pharmaceutical composition comprising an HCV inhibitory amount of at    least one of the following compounds:

or a pharmaceutically acceptable salt thereof together with apharmaceutically acceptable excipient.

-   Embodiment 53. A method for treating a subject for a Hepatitis C    viral (HCV) infection comprising administering to said subject a    pharmaceutical composition comprising an HCV inhibitory amount of at    least one of the following compounds:

or a pharmaceutically acceptable salt thereof together with apharmaceutically acceptable excipient.

-   Embodiment 54. The method of Embodiment 53, wherein said method    further comprises administering an additional anti-HCV agent    selected from the group consisting of pegylated interferon,    un-pegylated interferon, ribavirin or prodrugs or derivatives    thereof, a glucosidase inhibitor, a protease inhibitor, a polymerase    inhibitor, p7 inhibitors, an entry inhibitor, a fusion inhibitor, an    anti-fibrotic, a caspase inhibitor, a drug which targets inosine    monophosphate dehydrogenase inhibitors (IMPDH), synthetic thymosin    alpha 1, therapeutic vaccines, immunomodulators, a helicase    inhibitor, and a Toll-like receptor agonist.-   Embodiment 55. A compound having one of the following formulas:

-   Embodiment 56. A compound having one of the following formulas:

-   Embodiment 57. A method for treating or preventing infection by a    virus in a subject, wherein said virus comprises an internal    ribosome entry site (IRES), comprising administering to said subject    a pharmaceutical composition comprising a viral inhibitory amount of    at least one compound having the following formula:

wherein:

-   X is:-   hydrogen;-   a cyano group;-   an amino group;-   a

or

-   a 5- or 6-membered heteroaryl;-   a C₆ to C₈ aryl, optionally substituted with:

an alkoxy group,

a cyano group, or

a halogen;

-   or X together with Y forms:

-   Y is:-   a halogen;-   an amino group;-   a

or

-   a —SO₂R_(x), where R_(x) is a C₁ to C₆ alkyl;-   a cyano group;-   a —COOR_(x) group, where R_(x) is a C₁ to C₆ alkyl;-   a C₆ to C₈ aryl, optionally substituted with:

an alkoxy; or

a cyano group;

-   a —COR_(a) group, where R_(a) is:

an amino optionally substituted with one or two C₁ to C₆ alkyls, wherethe alkyls are optionally substituted with a C₆ to C₈ aryl;

a —NHR_(b) group where R_(b) is:

-   -   a C₆ to C₈ aryl optionally substituted with:        -   a haloalkyl; or        -   a halogen        -   a haloalkoxy; or

a 5- or 6-membered heterocycle optionally substituted with a C₁ to C₆alkyl;

a C₁ to C₆ alkyl;

a —SR_(x) group, where R_(x) is as defined above;

-   a 5 or 6 membered heteroaryl optionally substituted with:

a C₆ to C₈ aryl optionally substituted with:

-   -   an alkoxy    -   a halogen; or    -   a C₁ to C₆ alkyl;

a 5- or 6-membered heteroaryl optionally substituted with

-   -   an alkoxy    -   a halogen; or    -   a C₁ to C₆ alkyl;

-   a C₁ to C₆ alkyl, optionally substituted with a —OR_(c), where R_(c)    is a C₆ to C₈ aryl optionally substituted with one or more halogens;    or

-   a nitro group;

-   a —NHR_(d) group, where R_(d) is a C₆ to C₈ aryl optionally    substituted with an alkoxy

-   a —NHCOR_(e) group where R_(e) is:

a C₆ to C₈ aryl optionally substituted with a haloalkyl;

a C₁ to C₆ alkyl;

-   or together with X forms:

-   R is:-   a hydrogen-   a haloalkyl;-   a C₁ to C₆ alkyl optionally substituted with hydroxyl;-   a 5- or 6-member heteroaryl;-   a C₆ to C₈ aryl optionally substituted with one or more halogens;-   or R together with R₁ forms:

-   R₁ is:-   a hydrogen;-   a C₆ to C₈ aryl-   a C₁ to C₆ alkyl;-   a OCOR_(f) where R_(f) is a 5- or 6-membered heterocycle;-   an alkoxy optionally substituted with an amino group, wherein the    amino group is optionally substituted with one or two C₁ to C₆    alkyls, where the alkyls are optionally substituted with an amino    optionally substituted with one or two C₁ to C₆ alkyls-   an alkoxy optionally substituted with a 5 to 8 membered heterocycle    optionally substituted with a C₁ to C₆ alkyl, which is optionally    substituted with:

an alkoxy, or

an amino, optionally substituted with one or two C_(i) to C₆ alkyls;

-   or R₁ together with R₂ forms:

-   R₁ together with R forms:

-   R₂ is:-   a C₁ to C₆ alkyl;-   a 5 or 6-membered heterocycle;-   an amino optionally substituted with a C₁ to C₆ alkyl;-   or R₁ together with R₂ forms:

or a pharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable excipient.

-   Embodiment 58. The method of Embodiment 57, wherein said    pharmaceutical composition further comprises an additional    anti-viral agent.-   Embodiment 59. The method of Embodiment 58, wherein said additional    anti-viral agent is selected from the group consisting of pegylated    interferon, un-pegylated interferon, ribavirin or prodrugs or    derivatives thereof, a glucosidase inhibitor, a protease inhibitor,    a polymerase inhibitor, p7 inhibitors, an entry inhibitor, a fusion    inhibitor, an anti-fibrotic, a caspase inhibitor, a drug which    targets inosine monophosphate dehydrogenase inhibitors (IMPDH),    synthetic thymosin alpha 1, therapeutic vaccines, immunomodulators,    a helicase inhibitor, and a Toll-like receptor agonist.-   Embodiment 60. A method for treating or preventing infection by a    virus in a subject, wherein said virus comprises an internal    ribosome entry site (IRES), comprising administering to said subject    a pharmaceutical composition comprising a viral inhibitory amount of    at least one compound having the following formula:

wherein

-   X is amino or hydrogen;-   Y is-   a —COOR_(x) group, where R_(x) is as defined above;-   a —COR_(a) group, where R_(a) is:

an amino optionally substituted with one or two C₁ to C₆ alkyls, wherethe alkyls are optionally substituted with a C₆ to C₈ aryl;

a —SR_(x) group, where R_(x) is as defined above;

-   a 5 or 6 membered heteroaryl optionally substituted with:

a C₆ to C₈ aryl optionally substituted with:

-   -   an alkoxy    -   a halogen; or    -   a C₁ to C₆ alkyl;    -   a 5- or 6-membered heteroaryl optionally substituted with a C₆        to C₈ aryl optionally substituted with a halogen;

-   R is a C₁ to C₆ alkyl;

-   R₁ is a C₁ to C₆ alkyl or R₁ is selected from the group consisting    of

-   an alkoxy optionally substituted with an amino group, wherein the    amino group is optionally substituted with one or two C₁ to C₆    alkyls, where the alkyls are optionally substituted with an amino    optionally substituted with one or two C₁ to C₆ alkyls; and

-   an alkoxy optionally substituted with a 5 to 8 membered heterocycle    optionally substituted with a C₁ to C₆ alkyl, which is optionally    substituted with:

an amino, optionally substituted with one or two C₁ to C₆ alkyls; and

-   R₂ is—a C₁ to C₆ alkyl or an amino optionally substituted with a C₁    to C₆ alkyl;    or a pharmaceutically acceptable salt thereof, together with a    pharmaceutically acceptable excipient.-   Embodiment 61. The method of Embodiment 60, wherein said    pharmaceutical composition further comprises an additional    anti-viral agent.-   Embodiment 62. The method of Embodiment 61, wherein said additional    anti-viral agent is selected from the group consisting of pegylated    interferon, un-pegylated interferon, ribavirin or prodrugs or    derivatives thereof, a glucosidase inhibitor, a protease inhibitor,    a polymerase inhibitor, p7 inhibitors, an entry inhibitor, a fusion    inhibitor, an anti-fibrotic, a caspase inhibitor, a drug which    targets inosine monophosphate dehydrogenase inhibitors (IMPDH),    synthetic thymosin alpha 1, therapeutic vaccines, immunomodulators,    a helicase inhibitor, and a Toll-like receptor agonist.-   Embodiment 63. A pharmaceutical composition for affecting viral IRES    activity in a subject infected with a virus, comprising at least one    compound having the following formula:

wherein:

-   X is:-   hydrogen;-   a cyano group;-   an amino group;-   a

or

-   a 5- or 6-membered heteroaryl;-   a C₆ to C₈ aryl, optionally substituted with:

an alkoxy group,

a cyano group, or

a halogen;

-   or X together with Y forms:

-   Y is:-   a halogen;-   an amino group;-   a

or

-   a —SO₂R_(x), where R_(x) is a C₁ to C₆ alkyl;-   a cyano group;-   a —COOR_(x) group, where R_(x) is a C₁ to C₆ alkyl;-   a C₆ to C₈ aryl, optionally substituted with:

an alkoxy; or

a cyano group;

-   a —COR_(a) group, where R_(a) is:

an amino optionally substituted with one or two C₁ to C₆ alkyls, wherethe alkyls are optionally substituted with a C₆ to C₈ aryl

a —NHR_(b) group where R_(b) is:

-   -   a C₆ to C₈ aryl optionally substituted with:        -   a haloalkyl; or        -   a halogen        -   a haloalkoxy; or

a 5- or 6-membered heterocycle optionally substituted with a C₁ to C₆alkyl;

a C₁ to C₆ alkyl;

a —SR_(x) group, where R_(x) is as defined above;

-   a 5 or 6 membered heteroaryl optionally substituted with:

a C₆ to C₈ aryl optionally substituted with:

-   -   an alkoxy    -   a halogen; or    -   a C₁ to C₆ alkyl;

a 5- or 6-membered heteroaryl optionally substituted with

-   -   an alkoxy    -   a halogen; or    -   a C₁ to C₆ alkyl;

-   a C₁ to C₆ alkyl, optionally substituted with a —OR_(c), where R_(c)    is a C₆ to C₈ aryl optionally substituted with one or more    halogens;or

-   a nitro group;

-   a —NHR_(d) group, where R_(d) is a C₆ to C₈ aryl optionally    substituted with an alkoxy

-   a —NHCOR_(e) group where R_(e) is:

a C₆ to C₈ aryl optionally substituted with a haloalkyl;

a C₁ to C₆ alkyl;

-   or together with X forms:

-   R is:-   a hydrogen-   a haloalkyl;-   a C₁ to C₆ alkyl optionally substituted with hydroxyl;-   a 5- or 6-member heteroaryl;-   a C₆ to C₈ aryl optionally substituted with one or more halogens;-   or R together with R₁ forms:

-   R₁ is:-   a hydrogen;-   a C₆ to C₈ aryl-   a C₁ to C₆ alkyl;-   a OCOR_(f) where R_(f) is a 5- or 6-membered heterocycle;-   an alkoxy optionally substituted with an amino group, wherein the    amino group is optionally substituted with one or two C₁ to C₆    alkyls, where the alkyls are optionally substituted with an amino    optionally substituted with one or two C₁ to C₆ alkyls-   an alkoxy optionally substituted with a 5 to 8 membered heterocycle    optionally substituted with a C₁ to C₆ alkyl, which is optionally    substituted with:

an alkoxy, or

an amino, optionally substituted with one or two C₁ to C₆ alkyls;

-   or R₁ together with R₂ forms:

-   R₁ together with R forms:

-   R₂ is:-   a C₁ to C₆ alkyl;-   a 5 or 6-membered heterocycle;-   an amino optionally substituted with a C₁ to C₆ alkyl;-   or R₁ together with R₂ forms:

or a pharmaceutically acceptable salt thereof, together with a compoundknown in the art to affect IRES activity and a pharmaceuticallyacceptable excipient.

-   Embodiment 64. The pharmaceutical composition of Embodiment 50,    wherein said compound known in the art to affect IRES activity    affects IRES mediated translation of the viral proteins.-   Embodiment 65. A pharmaceutical composition for affecting viral IRES    activity in a subject infected with a virus, comprising at least one    compound having the following formula:

wherein

-   X is amino or hydrogen;-   Y is-   a —COOR_(x) group, where R_(x) is as defined above;-   a —COR_(a) group, where R_(a) is:

an amino optionally substituted with one or two C₁ to C₆ alkyls, wherethe alkyls are optionally substituted with a C₆ to C₈ aryl;

a —SR_(x) group, where R_(x) is as defined above;

-   a 5 or 6 membered heteroaryl optionally substituted with:

a C₆ to C₈ aryl optionally substituted with:

-   -   an alkoxy    -   a halogen; or    -   a C₁ to C₆ alkyl;    -   a 5- or 6-membered heteroaryl optionally substituted with a C₆        to C₈ aryl optionally substituted with a halogen;

-   R is a C₁ to C₆ alkyl;

-   R₁ is a C₁ to C₆ alkyl or R₁ is selected from the group consisting    of

-   an alkoxy optionally substituted with an amino group, wherein the    amino group is optionally substituted with one or two C₁ to C₆    alkyls, where the alkyls are optionally substituted with an amino    optionally substituted with one or two C₁ to C₆ alkyls; and

-   an alkoxy optionally substituted with a 5 to 8 membered heterocycle    optionally substituted with a C₁ to C₆ alkyl, which is optionally    substituted with:

an amino, optionally substituted with one or two C₁ to C₆ alkyls; and

-   R₂ is—a C₁ to C₆ alkyl or an amino optionally substituted with a C₁    to C₆ alkyl;    or a pharmaceutically acceptable salt thereof, together with a    compound known in the art to affect IRES activity and a    pharmaceutically acceptable excipient.-   Embodiment 66. The pharmaceutical composition of Embodiment 65,    wherein said compound known in the art to affect IRES activity    affects IRES mediated translation of the viral proteins.-   Embodiment 67. A method for affecting viral IRES activity in a    subject infected with a virus, comprising administering to said    subject at least one compound having the following formula:

wherein:

-   X is:-   hydrogen;-   a cyano group;-   an amino group;-   a

or

-   a 5- or 6-membered heteroaryl;-   a C₆ to C₈ aryl, optionally substituted with:

an alkoxy group,

a cyano group, or

a halogen;

or X together with Y forms:

-   Y is:-   a halogen;-   an amino group;-   a

or

-   a —SO₂R_(x), where R_(x) is a C₁ to C₆ alkyl;-   a cyano group;-   a —COOR_(x) group, where R_(x) is a C₁ to C₆ alkyl;-   a C₆ to C₈ aryl, optionally substituted with:

an alkoxy; or

a cyano group;

-   a —COR_(a) group, where R_(a) is:

an amino optionally substituted with one or two C₁ to C₆ alkyls, wherethe alkyls are optionally substituted with a C₆ to C₈ aryl;

a —NHR_(b) group where R_(b) is:

-   -   a C₆ to C₈ aryl optionally substituted with:        -   a haloalkyl; or        -   a halogen        -   a haloalkoxy; or

a 5- or 6-membered heterocycle optionally substituted with a C₁ to C₆alkyl;

a C₁ to C₆ alkyl;

a —SR_(x) group, where R_(x) is as defined above;

-   a 5 or 6 membered heteroaryl optionally substituted with:

a C₆ to C₈ aryl optionally substituted with:

-   -   an alkoxy    -   a halogen; or    -   a C₁ to C₆ alkyl;

a 5- or 6-membered heteroaryl optionally substituted with

-   -   an alkoxy    -   a halogen; or    -   a C₁ to C₆ alkyl; a C₁ to C₆ alkyl, optionally substituted with        a —OR_(c), where R_(c) is a C₆ to C₈ aryl optionally substituted        with one or more halogens; or

-   a nitro group;

-   a —NHR_(d) group, where R_(d) is a C₆ to C₈ aryl optionally    substituted with an alkoxy

-   a —NHCOR_(e) group where R_(e) is:

a C₆ to C₈ aryl optionally substituted with a haloalkyl;

a C₁ to C₆ alkyl

-   or together with X forms:

-   R is:-   a hydrogen-   a haloalkyl;-   a C₁ to C₆ alkyl optionally substituted with hydroxyl;-   a 5- or 6-member heteroaryl;-   a C₆ to C₈ aryl optionally substituted with one or more halogens;-   or R together with R₁ forms:

-   R₁ is:-   a hydrogen;-   a C₆ to C₈ aryl-   a C₁ to C₆ alkyl;-   a OCOR_(f) where R_(f) is a 5- or 6-membered heterocycle;-   an alkoxy optionally substituted with an amino group, wherein the    amino group is optionally substituted with one or two C₁ to C₆    alkyls, where the alkyls are optionally substituted with an amino    optionally substituted with one or two C₁ to C₆ alkyls-   an alkoxy optionally substituted with a 5 to 8 membered heterocycle    optionally substituted with a C₁ to C₆ alkyl, which is optionally    substituted with:

an alkoxy, or

an amino, optionally substituted with one or two C₁ to C₆ alkyls;

-   or R₁ together with R₂ forms:

-   R₁ together with R forms:

-   R₂ is:-   a C₁ to C₆ alkyl;-   a 5 or 6-membered heterocycle;-   an amino optionally substituted with a C₁ to C₆ alkyl;-   or R₁ together with R₂ forms:

or a pharmaceutically acceptable salt thereof, together with apharmaceutically acceptable excipient.

-   Embodiment 68. A method for affecting viral IRES activity in a    subject infected with a virus, comprising administering to said    subject at least one compound having the following formula:

wherein

-   X is amino or hydrogen;-   Y is-   a —COOR_(x) group, where R_(x) is as defined above;-   a —COR_(a) group, where R_(a) is:

an amino optionally substituted with one or two C₁ to C₆ alkyls, wherethe alkyls are optionally substituted with a C₆ to C₈ aryl;

a —SR_(x) group, where R_(x) is as defined above;

-   a 5 or 6 membered heteroaryl optionally substituted with:

a C₆ to C₈ aryl optionally substituted with:

-   -   an alkoxy    -   a halogen; or    -   a C₁ to C₆ alkyl;    -   a 5- or 6-membered heteroaryl optionally substituted with a C₆        to C₈ aryl optionally substituted with a halogen;

-   R is a C₁ to C₆ alkyl;

-   R₁ is a C₁ to C₆ alkyl or R₁ is selected from the group consisting    of

-   an alkoxy optionally substituted with an amino group, wherein the    amino group is optionally substituted with one or two C₁ to C₆    alkyls, where the alkyls are optionally substituted with an amino    optionally substituted with one or two C₁ to C₆ alkyls; and

-   an alkoxy optionally substituted with a 5 to 8 membered heterocycle    optionally substituted with a C₁ to C₆ alkyl, which is optionally    substituted with:

an amino, optionally substituted with one or two C₁ to C₆ alkyls; and

-   R₂ is—a C₁ to C₆ alkyl or an amino optionally substituted with a C₁    to C₆ alkyl;    or a pharmaceutically acceptable salt thereof, together with a    pharmaceutically acceptable excipient.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 illustrates the HCV-PV chimera construct. The cloverleaf-like RNAstructure of PV, an essential cis-acting replication signal ending withthe genome-linked protein VPg, is located at the 5′ end of the genome.The solid (HCV) and open (PV) boxes depict open reading frames encodingviral polypeptides. The position of the HCV core fragment (the first 123amino acids) gene is denoted by Δ Core. Overall, the HCV specificsequence in the HCV-PV spans from nucleotides 18 to 710. (139).

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, compounds that modify HCVtranslation have been identified and methods of using these compoundsfor preventing or treating HCV infection are provided. Without beinglimited to one theory, it is believed that the compounds of the presentinvention inhibit IRES-mediated initiation and translation. The HCV IRESdirects the translation of the viral polyprotein that isposttranslationally processed into at least 10 mature viral proteins,including the structural proteins core (putative nucleocapsid), E1 andE2 and the nonstructural (NS) proteins NS2 to NS5B.

A. Compounds of the Invention

In one aspect of the invention, compounds of the invention are providedwhich may be useful for preventing or treating HCV infection.

Preferred compounds of the present invention useful in the inhibition ofHCV-IRES mediated initiation and translation include those of Formula(I) as shown below.

wherein:

-   X is:-   hydrogen;-   a cyano group;-   an amino group;-   a

or

-   a 5- or 6-membered heteroaryl;-   a C₆ to C₈ aryl, optionally substituted with:

an alkoxy group,

a cyano group, or

a halogen;

or X together with Y forms:

-   Y is:-   a halogen;-   an amino group;-   a

or

-   a —SO₂R_(x), where R_(x) is a C₁ to C₆ alkyl;-   a cyano group;-   a —COOR_(x) group, where R_(x) is a C₁ to C₆ alkyl;-   a C₆ to C₈ aryl, optionally substituted with:

an alkoxy; or

a cyano group;

-   a —COR_(a) group, where R_(a) is:

an amino optionally substituted with one or two C₁ to C₆ alkyls, wherethe alkyls are optionally substituted with a C₆ to C₈ aryl;

a —NHR_(b) group where R_(b) is:

-   -   a C₆ to C₈ aryl optionally substituted with:        -   a haloalkyl; or        -   a halogen        -   a haloalkoxy; or

a 5- or 6-membered heterocycle optionally substituted with a C₁ to C₆alkyl;

a C₁ to C₆ alkyl;

a —SR_(x) group, where R_(x) is as defined above;

-   a 5 or 6 membered heteroaryl optionally substituted with:

a C₆ to C₈ aryl optionally substituted with:

-   -   an alkoxy    -   a halogen; or    -   a C₁ to C₆ alkyl;

a 5- or 6-membered heteroaryl optionally substituted with

-   -   an alkoxy    -   a halogen; or    -   a C₁ to C₆ alkyl;

-   a C₁ to C₆ alkyl, optionally substituted with a —OR_(c), where R_(c)    is a C₆ to C₈ aryl optionally substituted with one or more    halogens;or

-   a nitro group;

-   a —NHR_(d) group, where R_(d) is a C₆ to C₈ aryl optionally    substituted with an alkoxy

-   a —NHCOR_(e) group where R_(e) is:

a C₆ to C₈ aryl optionally substituted with a haloalkyl;

a C₁ to C₆ alkyl;

-   or together with X forms:

-   R is:-   a hydrogen-   a haloalkyl;-   a C₁ to C₆ alkyl optionally substituted with hydroxyl;-   a 5- or 6-member heteroaryl;-   a C₆ to C₈ aryl optionally substituted with one or more halogens;-   or R together with R₁ forms:

-   R₁ is:-   a hydrogen;-   a C₆ to C₈ aryl-   a C₁ to C₆ alkyl;-   a OCOR_(f) where R_(f) is a 5- or 6-membered heterocycle;-   an alkoxy optionally substituted with an amino group, wherein the    amino group is optionally substituted with one or two C₁ to C₆    alkyls, where the alkyls are optionally substituted with an amino    optionally substituted with one or two C₁ to C₆ alkyls-   an alkoxy optionally substituted with a 5 to 8 membered heterocycle    optionally substituted with a C₁ to C₆ alkyl, which is optionally    substituted with:

an alkoxy, or

an amino, optionally substituted with one or two C₁ to C₆ alkyls;

-   or R₁ together with R₂ forms:

-   R₁ together with R forms:

-   R₂ is:-   a C₁ to C₆ alkyl;-   a 5 or 6-membered heterocycle;-   an amino optionally substituted with a C₁ to C₆ alkyl;-   or R₁ together with R₂ forms:

or a pharmaceutically acceptable salt thereof, together with anadditional anti-HCV agent and a pharmaceutically acceptable excipient.

In another preferred embodiment, a compound or a composition of thepresent invention includes a compound of Formula I, wherein the compoundof Formula I is not

As used herein, the term “alkyl” generally refers to saturatedhydrocarbyl radicals of straight, branched or cyclic configuration, orcombinations of cyclic and branched or straight, including methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl,n-pentyl, n-hexyl, cyclohexyl, n-heptyl, octyl, n-octyl, and the like.In some embodiments, alkyl substituents may be C₁ to C₈ or C₁ to C₆alkyl groups.

As used herein, “alkylene” generally refers to linear, branched orcyclic alkene radicals having one or more carbon-carbon double bonds,such as C₂ to C₆ alkylene groups including 3-propenyl.

As used herein, “aryl” refers to a carbocyclic aromatic ring structure.Included in the scope of aryl groups are aromatic rings having from fiveto twenty carbon atoms. Aryl ring structures include compounds havingone or more ring structures, such as mono-, bi-, or tricyclic compounds.Examples of aryl groups that include phenyl, tolyl, anthracenyl,fluorenyl, indenyl, azulenyl, phenanthrenyl (i.e., phenanthrene), andnapthyl (i.e., napthalene) ring structures. In certain embodiments, thearyl group may be optionally substituted.

As used herein, “heteroaryl” refers to cyclic aromatic ring structuresin which one or more atoms in the ring, the heteroatom(s), is an elementother than carbon. Heteroatoms are typically O, S or N atoms. Includedwithin the scope of heteroaryl, and independently selectable, are O, N,and S heteroaryl ring structures. The ring structure may includecompounds having one or more ring structures, such as mono-, bi-, ortricyclic compounds. In some embodiments, the heteroaryl groups may beselected from heteroaryl groups that contain two or more heteroatoms,three or more heteroatoms, or four or more heteroatoms. Heteroaryl ringstructures may be selected from those that contain five or more atoms,six or more atoms, or eight or more atoms. Examples of heteroaryl ringstructures include: acridine, benzimidazole, benzoxazole, benzodioxole,benzofuran, 1,3-diazine, 1,2-diazine, 1,2-diazole, 1,4-diazanaphthalene,furan, furazan, imidazole, indole, isoxazole, isoquinoline, isothiazole,oxazole, purine, pyridazine, pyrazole, pyridine, pyrazine, pyrimidine,pyrrole, quinoline, quinoxaline, thiazole, thiophene, 1,3,5-triazine,1,2,4-triazine, 1,2,3-triazine, tetrazole and quinazoline.

As used herein, “heterocycle” refers to cyclic ring structures in whichone or more atoms in the ring, the heteroatom(s), is an element otherthan carbon. Heteroatoms are typically O, S or N atoms. Included withinthe scope of heterocycle, and independently selectable, are O, N, and Sheterocycle ring structures. The ring structure may include compoundshaving one or more ring structures, such as mono-, bi-, or tricycliccompounds. Example of heterocyclo groups include morpholinyl,pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperazinyl, hydantoinyl,valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl or tetrahydrothiopyranyl and the like. In certainembodiments, the heterocycle may optionally be substituted.

As used herein, “alkoxy” generally refers to a group with the structure—O—R. In certain embodiments, R may be an alkyl group, such as a C₁ toC₈ alkyl group.

For the purposes of this invention, halo substituents may beindependently selected from the halogens such as fluorine, chlorine,bromine, iodine, and astatine. A haloalkyl is an alkyl group, as definedabove, substituted with one or more halogens. A haloalkoxy is an alkoxygroup, as defined above, substituted with one or more halogens.

For the purposes of this invention, where one or more functionalitiesencompassing X, Y, R, R₁ and R₂ are incorporated into a molecule ofFormula (I), each functionality appearing at any location within thedisclosed molecule may be independently selected, and as appropriate,independently substituted. Further, where a more generic substituent isset forth for any position in the molecules of the present invention, itis understood that the generic substituent may be replaced with morespecific substituents, and the resulting molecules are within the scopeof the molecules of the present invention.

By “substituted” or “optionally substituted” it is meant that theparticular substituent may be substituted with a chemical group known toone of skill in the art to be appropriate for the referred tosubstituent, unless a chemical group is specifically mentioned.

Exemplary X substituents include the following, where the * indicatesthe bond of attachment of the scaffold molecule.

Preferred X substituents include an amino group or a hydrogen.Particularly preferred X substituents include an amino group.

Exemplary Y substituents include the following, where the * indicatesthe bond of attachment of the scaffold molecule.

Preferred Y substituents include the following, where the * indicatesthe bond of attachment of the scaffold molecule.

Exemplary R substituents include the following, where the * indicatesthe bond of attachment of the scaffold molecule.

Preferred R substituents include C₁ to C₆ alkyl and more preferred Rsubstituents include methyl.

Exemplary R₁ substituents include the following, where the * indicatesthe bond of attachment of the scaffold molecule.

Preferred R₁ substituents include the following, where the * indicatesthe bond of attachment of the scaffold molecule.

Exemplary R₂ substituents include the following, where the * indicatesthe bond of attachment of the scaffold molecule.

Preferred R₂ substituents include the following, where the * indicatesthe bond of attachment of the scaffold molecule.

Compounds of the invention include the following:

The above compounds were prepared using the schemes and examples setforth below. Other methods of producing these compounds are known to oneof skill in the art.

Preferred compounds of the present invention include the following:

B. Preparation of Compounds of the Invention

Compounds of the invention may be produced in any manner known in theart. By way of example, compounds of the invention may be preparedaccording to the following general schemes.

Thienopyridine compounds of the present invention can be obtained viastandard, well known synthetic methodology. All the starting materialsand intermediates for preparing the compounds in the invention arecommercially available or can be prepared from commercially availablematerials using known synthetic methods and reagents.

Compounds of formula I, represented by structure II, where R₃ representsan electron deficient group, such as aryl, heteroaryl, cyano, COOR_(c),COR_(d), CONR_(a)R_(b), NO₂, CONR_(a)SO₂R_(e), SO₂R_(e) andSO₂NR_(a)R_(b), can be prepared using the method depicted in Scheme A.

Treatment of 1,3-diketones A1 with 2-cyanothioacetamide A2 in a suitablesolvent, such as alcohol or a polar aprotic solvent, in the presence ofan organic or inorganic base, such as triethylamine at a temperaturefrom ambient to 80° C. gives the intermediate 2-mercapto-3-cyanopyridineA3. A3 can be treated directly with A4, where L represents a suitableleaving group, which is attached to an activated methylene, in thepresence of a base, such as sodium methoxide (NaOMe), to give compoundsof formula II.

Preferably, A3 can be isolated using a standard aqueous work-upprocedure, then treated with A4 in a polar solvent, such asdimethylformamide (DMF), in the presence of a base, such as potassiumcarbonate (K₂CO₃), at an elevated temperature, e.g., 50-90° C., to givecompounds of formula II. Sodium hydride can also be deployed for thetransformation of less reactive substrates.

Alternatively, compounds of formula I, represented by structure II canbe prepared using the method depicted in Scheme B.

Condensation of aldehydes B1 and 2-cyanothioacetamide B2 in a suitablesolvent, such as alcohol, in the presence of aluminium oxide gives theintermediate B3. Condensation of B3 with ketones B4 in the presence of abase, e.g., piperidine or potassium hexamethyldisilazane (KHMDS),followed by ring-closure and self-oxidation gives the intermediates,2-mercapto-3-cyanopyridines B5. Conversion of B5 to compounds of formulaII can be realized by the treatment of B5 with B6 using the methoddescribed previously.

Compounds of formula I, represented by structure III can also beprepared using the method depicted in Scheme C.

Treatment of ketoenamines Cl with 2-cyanothioacetamide C2 in a suitablesolvent, such as alcohol or a polar aprotic solvent, in the presence ofan organic or inorganic base, such as triethylamine at a temperaturefrom ambient to 80° C. gives the intermediate2-mercapto-3-cyanopyridines C3. Compounds C3 can then be converted toformula III compounds using the method described previously.

Compounds of formula I, represented by structure IV can be preparedusing the method depicted in Scheme D.

5-Thienopyridinyl acetate D1, prepared using the method depicted inSchemes A and B (where R₁ 50 OAc), can be converted to D2 by a standardbasic hydrolysis. Compounds D2 can be treated with 1,2-dihaloethane D3,such as 1-bromo-2-chloroethane, in a suitable solvent such asacetonitrile, in the presence of a base, eg., potassium carbonate, at anelevated temperature, typically 50˜90° C., to compounds D4. Treatment ofD4 with a primary or a secondary amine D5, in a suitable solvent in thepresence of a base gives compounds of formula IV.

Compounds of formula I, represented by structure V can be prepared usingthe method depicted in Scheme E.

5-Hydroxythienopyridine E1, prepared using the method depicted in SchemeD, can be acetylated with compounds of type E2 in the presence of a baseto give compounds of formula V.

Compounds of formula I, represented by structure VI can be preparedusing the method depicted in Scheme F.

Compound F1 can be treated with trifluoromethylsulfonic anhydride in asuitable solvent such as dichloromethane in the presence of a base, eg.,4-dimethylaminopyridine, to give triflate F2. Treatment of F2 witharylboronic acids F3 using Suzuki coupling methodology in the presenceof a palladium catalyst gives compounds of formula VI.

Compounds of formula I, represented by structure VII can be preparedusing the method depicted in Scheme G.

Thienopyridines of type G3, where L represents a suitable leaving group,preferably halogen, such as Cl and Br, can be prepared by the treatmentof G1 with G2, in the presence of a base. Treatment of G3, with avariety of amines G4 at an elevated temperature gives compounds offormula VII.

Compounds of formula I, represented by structure VIII can be preparedusing the method depicted in Scheme H.

Treatment of 3-aminothienopyridines H1, prepared using the methoddepicted in Schemes A, or B, with 2,5-dimethoxytetrahydrofuran in thepresence of an inorganic or organic acid, such as acetic acid, in asuitable solvent, or use of acetic acid as the solvent at a temperaturefrom 20 to 90° C., gives compounds of formula VIII.

Compounds of formula I, represented by structure IX can be preparedusing the method depicted in Scheme I.

3-Aminothienopyridines I1, prepared using the method depicted in SchemesA or B, can be converted to 3-iodothienopyridines I2 with KI or CuI, viaa diazonium salt intermediate, generated in situ using standarddiazotization methods, such as sodium nitrite in aqueous acid media, orusing an organic nitrite, such as BuONO in a suitable solvent, such asacetonitrile. The iodide I2 can then be treated with CuCN in a suitablesolvent, e.g., N-methylpyrrolidinone, at temperature of 25-180° C. togive the compounds of formula IX.

Compounds of formula I, represented by structure X can be prepared usingthe method depicted in Scheme J.

3-iodothienopyridines J1, prepared using the method depicted in SchemeI, can be reacted with aryl or heteroarylboronic acids J2 in thepresence of a palladium catalyst under standard Suzuki couplingcondition to give the compounds of formula X.

Compounds of formula I, represented by structure XI, can be preparedusing the method depicted in Scheme K.

Thienopyridine-2-carboxylates K1, prepared using methods describedpreviously, can be converted to the corresponding acids K2 usingstandard hydrolysis or de-alkylation methods. For example, K1 can betreated with sodium hydroxide solution in a suitable solvent, such asTHF at an elevated temperature from 50-80° C., to give K2.Alternatively, K2 can be obtained from K1 using a selectivede-alkylation method, such as standard trifluoroacetic acidde-t-butylation, e.g., when K1 is a t-butyl ester; or standardde-methylation using BBr₃ or trimethylsilyl iodide, e.g., when K1 is amethyl ester. K2 then can be activated as an acid chloride by thetreatment of the acids with thionyl chloride or oxalyl chloride, oralternatively, activated as an active ester or active anhydride usingstandard peptide coupling chemistry, for example, PyBOP in DMF, followedby the treatment with amines K3 to give the compounds of formula XI.

Compounds of formula I, represented by structure XII, where R₇represents phenyl optionally substituted with a C1 to C6 alkyl or a 5 or6 membered heteroaryl optionally substituted with a C1 to C6 alkyl, canbe prepared using the method depicted in Scheme L.

Thienopyridine-2-carboxylic acids L1, prepared using methods depicted inprevious Schemes, can be activated with DCC or PS-carbodiimide in asuitable solvent system, such as dichloroethane-DMF, followed by thetreatment with tetrazoles L2. Heating the reaction at an elevatedtemperature, e.g., 50-150° C. gives the compounds of formula XII.

Compounds of formula I, represented by structure XIII, can be preparedusing the method depicted in Scheme M.

Hydroxyamidines of type M1, commercially available, can be reacted with2-chloroacetyl chloride M2 in a suitable aprotic solvent, such asdichloromethane, in the presence of a base, e.g. PS-DIEA, to giveO-acylated hydroxyamines M3. Heating M3 in a suitable solvent attemperature from 80-110° C. gives 5-chloromethyl-1,2,4-oxadiazoles oftype M4. Treatment of M4 with M5, which can be obtained using thechemistry described in Scheme A, in a suitable solvent, preferably polarprotic, or aprotic solvent, such as MeOH or DMF in the presence of abase, e.g. NaOMe or K₂CO₃ at an elevated temperature 50-80° C. to givethe compounds of formula XIII.

Compounds of formula I, represented by structure XIV, can be preparedusing the method depicted in Scheme N.

Thienopyridine-2-carboxylic acids N1, prepared using the method depictedin the previous Schemes, upon reaction with diphenylphosphoryl azide(DPPA) (N2) in the presence of an organic base, such as triethylamine intert-butanol undergo Curtis rearrangement at an elevated temperature, togive N3, which can be converted to 2-amino thienopyridines in 50% TFA indichloromethane, to give the compounds of formula XIV.

Compounds of formula I, represented by structure XV, can be preparedusing the method depicted in Scheme O.

3-Amino-2-methylsulfonylthienopyridines O1, prepared using the methoddescribed previously in Scheme A, can be converted to3-iodo-2-methylsulfonylthienopyridines O2 by the treatment of O1 withbutylnitrite and CuI in acetonitrile at an elevated temperature, forexample, 50-70° C. Treatment of O2 with CuCN in NMP at temperaturesbetween ambient and 60° C. gives 3-cyano derivatives O3. The2-methylsulfonyl group of O3 can then be reacted with amines of type O4in a suitable solvent, such as acetonitrile at a temperature between25-85° C. to give compounds of structure XV.

Compounds of formula I, represented by structure XVI, can be preparedusing the method depicted in Scheme P.

2-Aminothienopyridines P1, prepared using the methods describedpreviously, can be treated with an acid chloride (P2) in the presence ofa base, such as triethyl amine, in a suitable organic solvent, such asdichloromethane, or pyridine as the base and the solvent at temperaturefrom ambient to 110° C. to give compounds of structure XVI.

Compounds of formula I, represented by structures XVII can be preparedusing the method depicted in Scheme Q.

2-Aminothienopyridines Q1, prepared using the method describedpreviously can be converted to 2-halothienopyridines Q3, where halogenis preferably, bromo or iodo, using standard Sandmeyer diazoniumchemistry, by the treatment of Q1 with NaNO₂ in aqueous acidic media,such as concentrated HCl, followed by the addition of copper halide Q2.Alternatively, Q3 can also be obtained by the treatment of Q1 with anorganic nitrite, such as butylnitrite in the presence of copper halidein a suitable solvent, preferably, acetonitrile at an elevatedtemperature from 50-70° C. Compounds Q3 can then be subjected to Suzukitype coupling using a palladium catalyst with various aryl- orheteroarylboronic acids Q4, in a suitable solvent system to givecompounds of structure XVII.

Compounds of formula I, represented by structures XVIII can be preparedusing the method depicted in Scheme R.

2-Halothienopyridines R1, prepared using the method described in theprevious Scheme S, where halogen is preferably, chloro, bromo or iodo,can be subjected to palladium catalyzed amination reaction with varioussubstituted anillines (R2), in the presence of a base, such as potassiumtert-butoxide, in a suitable solvent system, such as toluene, to givecompounds of structure XVIII. Alternatively, XVIII can be prepared byadopting the similar chemistry from R3, which can be synthesized usingthe chemistry described in scheme P and halo-aryl compounds, R4.

Compounds of formula I, represented by structures XIX can be preparedusing the method depicted in Scheme S.

Treatment of compounds of type S1 can then be reacted with an arylaldehyde (S2), for example benzaldehyde, in refluxing acetic acid toprovide compounds of structure XIX.

C. Methods of the Invention

The methods of the invention generally comprise administering atherapeutically effective amount of at least one compound of the presentinvention to a subject in need of treatment for HCV infection. In apreferred embodiment, a therapeutically effective amount of acomposition comprising a compound of Formula I as described herein isadministered to a subject in need of treatment. In another preferredembodiment, a compound or a composition used in the methods of thepresent invention includes a compound of Formula I as described hereinwherein the compound of Formula I is not

In another preferred embodiment, a compound or a composition of thepresent invention comprises a compound of Formula I, wherein thecompound of Formula I is not

The compound(s) of the present invention may be administered to thesubject via any drug delivery route known in the art. Specific exemplaryadministration routes include oral, ocular, rectal, buccal, topical,nasal, ophthalmic, subcutaneous, intramuscular, intraveneous (bolus andinfusion), intracerebral, transdermal, and pulmonary. Individualsinfected with HCV can be treated with the compounds of the presentinvention to prevent or reduce further replication of HCV.

The term therapeutically effective amount, as used herein, refers to anamount of a compound of the present invention effective to inhibit HCVtranslation, thereby effectively treating or ameliorating the HCVinfection. The effect of the compound can be determined by analyzing (1)the presence of HCV-RNA; (2) the presence of anti-HCV antibodies; (3)the level of serum alanine amino transferase (ALT) and aspartateaminotransferase (AST) (ALT and AST are elevated in patients chronicallyinfected with HCV); or (4) hepatocellular damage or any combinationthereof. The precise effective amount for a subject will depend upon thesubject's body weight, size and health. Therapeutically effectiveamounts for a given patient can be determined by routine experimentationthat is within the skill and judgment of the clinician.

For any compound, the therapeutically effective amount can be estimatedinitially either in cell culture assays or in relevant animal models,such as marmosets and tamarins. The animal model may also be used todetermine the appropriate concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans. Therapeutic efficacy andtoxicity may be determined by standard pharmaceutical procedures in cellcultures or experimental animals, e.g., ED₅₀ (the dose therapeuticallyeffective in 50% of the population) and LD₅₀ (the dose lethal to 50% ofthe population). The dose ratio between therapeutic and toxic effects isthe therapeutic index, and it can be expressed as the ratio, LD₅₀/ED₅₀.Pharmaceutical compositions that exhibit large therapeutic indices arepreferred. The dosage contained in such compositions is preferablywithin a range of circulating concentrations that include an ED₅₀ withlittle or no toxicity. The dosage may vary within this range dependingupon the dosage form employed, sensitivity of the patient, and the routeof administration.

More specifically, the concentration-biological effect relationshipsobserved with regard to the compound(s) of the present inventionindicate an initial target plasma concentration ranging fromapproximately 0.1 μg/ml to approximately 100 μg/mL, preferably fromapproximately 1 μg/mL to approximately 50 μg/mL, more preferably fromapproximately 5 μg/mL to approximately 50 μg/mL, even more preferablyfrom approximately 10 μg/mL to approximately 25 μg/mL. To achieve suchplasma concentrations, the compounds of the invention may beadministered at doses that vary from 0.1 μg to 100,000 mg, dependingupon the route of administration. Guidance as to particular dosages andmethods of delivery is provided in the literature and is generallyavailable to practitioners in the art. In general the dose will be inthe range of about 1 mg/day to about 10 g/day, or about 0.1 g to about 3g/day, or about 0.3 g to about 3 g/day, or about 0.5 g to about 2 g/day,in single, divided, or continuous doses for a patient weighing betweenabout 40 to about 100 kg (which dose may be adjusted for patients aboveor below this weight range, particularly children under 40 kg).

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activeagent(s) or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

D. Metabolites of the Compounds of the Invention

Also falling within the scope of the present invention are the in vivometabolic products of the compounds described herein. Such products mayresult for example from the oxidation, reduction, hydrolysis, amidation,esterification and the like of the administered compound, primarily dueto enzymatic processes. Accordingly, the invention includes compoundsproduced by a process comprising contacting a compound of this inventionwith a mammalian tissue or a mammal for a period of time sufficient toyield a metabolic product thereof. Such products typically areidentified by preparing a radio-labeled (e.g. C¹⁴ or H³) compound of theinvention, administering it in a detectable dose (e.g., greater thanabout 0.5 mg/kg) to a mammal such as rat, mouse, guinea pig, monkey, orto man, allowing sufficient time for metabolism to occur (typicallyabout 30 seconds to 30 hours), and isolating its conversion productsfrom urine, blood or other biological samples. These products are easilyisolated since they are labeled (others are isolated by the use ofantibodies capable of binding epitopes surviving in the metabolite). Themetabolite structures are determined in conventional fashion, e.g., byMS or NMR analysis. In general, analysis of metabolites may be done inthe same way as conventional drug metabolism studies well-known to thoseskilled in the art. The conversion products, so long as they are nototherwise found in vivo, are useful in diagnostic assays for therapeuticdosing of the compounds of the invention even if they possess nobiological activity of their own.

E. Pharmaceutical Compositions of the Invention

While it is possible for the compounds of the present invention to beadministered neat, it may be preferable to formulate the compounds aspharmaceutical compositions. As such, in yet another aspect of theinvention, pharmaceutical compositions useful in the methods of theinvention are provided. The pharmaceutical compositions of the inventionmay be formulated with pharmaceutically acceptable excipients such ascarriers, solvents, stabilizers, adjuvants, diluents, etc., dependingupon the particular mode of administration and dosage form. Thepharmaceutical compositions should generally be formulated to achieve aphysiologically compatible pH, and may range from a pH of about 3 to apH of about 11, preferably about pH 3 to about pH 7, depending on theformulation and route of administration. In alternative embodiments, itmay be preferred that the pH is adjusted to a range from about pH 5.0 toabout pH 8.0.

More particularly, the pharmaceutical compositions of the inventioncomprise a therapeutically or prophylactically effective amount of atleast one compound of the present invention, together with one or morepharmaceutically acceptable excipients. A therapeutically orprophylactically effective amount of a compound of the present inventionincludes a viral inhibitory amount of said compound. By “viralinhibitory amount” it is meant an amount sufficient to inhibit viralreplication or infectivity. Optionally, the pharmaceutical compositionsof the invention may comprise a combination of compounds of the presentinvention, or may include any additional active ingredient useful in thetreatment of viral infections, such as anti-viral agents that include,but are not limited to: pegylated interferon, including by way ofnon-limiting example alpha-interferon; un-pegylated interferon,including by way of non-limiting example alpha-interferon; ribavirin orprodrugs or derivatives thereof; a glucosidase inhibitor; proteaseinhibitors; polyermase inhibitors; p7 inhibitors; entry inhibitors,including fusion inhibitors such as Fuzeon™ (Trimeris); helicaseinhibitors; anti-fibrotics; caspase inhibitors; Toll-like receptoragonists; drugs that target IMPDH (inosine monophosphate dehydrogenaseinhibitors), such as Merimepodib™ (Vertex Pharmaceuticals Inc.);synthetic thymosin alpha 1 (ZADAXIN™, SciClone Pharmaceuticals Inc.);prophylactic vaccines, therapeutic viral vaccines, such as thoseproduced by Chiron, and therapeutic antibodies such as those produced byInnogenetics and XTL; and immunomodulators, such as histamine.

Formulations of the present invention, e.g., for parenteral or oraladministration, are most typically solids, liquid solutions, emulsionsor suspensions, while inhaleable formulations for pulmonaryadministration are generally liquids or powders, with powderformulations being generally preferred. A preferred pharmaceuticalcomposition of the invention may also be formulated as a lyophilizedsolid that is reconstituted with a physiologically compatible solventprior to administration. Alternative pharmaceutical compositions of theinvention may be formulated as syrups, creams, ointments, tablets, andthe like.

The term “pharmaceutically acceptable excipient” refers to an excipientfor administration of a pharmaceutical agent, such as the compounds ofthe present invention. The term refers to any pharmaceutical excipientthat may be administered without undue toxicity. Pharmaceuticallyacceptable excipients are determined in part by the particularcomposition being administered, as well as by the particular method usedto administer the composition. Accordingly, there exists a wide varietyof suitable formulations of pharmaceutical compositions of the presentinvention (see, e.g., Remington's Pharmaceutical Sciences).

Suitable excipients may be carrier molecules that include large, slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,and inactive virus particles. Other exemplary excipients includeantioxidants such as ascorbic acid; chelating agents such as EDTA;carbohydrates such as dextrin, hydroxyalkylcellulose,hydroxyalkylmethylcellulose, stearic acid; liquids such as oils, water,saline, glycerol and ethanol; wetting or emulsifying agents; pHbuffering substances; and the like. Liposomes are also included withinthe definition of pharmaceutically acceptable excipients.

The pharmaceutical compositions of the invention may be formulated inany form suitable for the intended method of administration. Whenintended for oral use for example, tablets, troches, lozenges, aqueousor oil suspensions, non-aqueous solutions, dispersible powders orgranules (including micronized particles or nanoparticles), emulsions,hard or soft capsules, syrups or elixirs may be prepared. Compositionsintended for oral use may be prepared according to any method known tothe art for the manufacture of pharmaceutical compositions, and suchcompositions may contain one or more agents including sweetening agents,flavoring agents, coloring agents and preserving agents, in order toprovide a palatable preparation.

Pharmaceutically acceptable excipients particularly suitable for use inconjunction with tablets include, for example, inert diluents, such ascelluloses, calcium or sodium carbonate, lactose, calcium or sodiumphosphate; disintegrating agents, such as croscarmellose sodium,cross-linked povidone, maize starch, or alginic acid; binding agents,such as povidone, starch, gelatin or acacia; and lubricating agents,such as magnesium stearate, stearic acid or talc. Tablets may beuncoated or may be coated by known techniques includingmicroencapsulation to delay disintegration and adsorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate alone or with a wax may be employed.

Formulations for oral use may be also presented as hard gelatin capsuleswhere the active ingredient is mixed with an inert solid diluent, forexample celluloses, lactose, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with non-aqueousor oil medium, such as glycerin, propylene glycol, polyethylene glycol,peanut oil, liquid paraffin or olive oil.

In another embodiment, pharmaceutical compositions of the invention maybe formulated as suspensions comprising a compound of the presentinvention in an admixture with at least one pharmaceutically acceptableexcipient suitable for the manufacture of a suspension. In yet anotherembodiment, pharmaceutical compositions of the invention may beformulated as dispersible powders and granules suitable for preparationof a suspension by the addition of suitable excipients.

Excipients suitable for use in connection with suspensions includesuspending agents, such as sodium carboxymethylcellulose,methylcellulose, hydroxypropyl methylcelluose, sodium alginate,polyvinylpyrrolidone, gum tragacanth, gum acacia, dispersing or wettingagents such as a naturally occurring phosphatide (e.g., lecithin), acondensation product of an alkylene oxide with a fatty acid (e.g.,polyoxyethylene stearate), a condensation product of ethylene oxide witha long chain aliphatic alcohol (e.g., heptadecaethyleneoxycethanol), acondensation product of ethylene oxide with a partial ester derived froma fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitanmonooleate); and thickening agents, such as carbomer, beeswax, hardparaffin or cetyl alcohol. The suspensions may also contain one or morepreservatives such as acetic acid, methyl and/or n-propylp-hydroxy-benzoate; one or more coloring agents; one or more flavoringagents; and one or more sweetening agents such as sucrose or saccharin.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, suchas olive oil or arachis oil, a mineral oil, such as liquid paraffin, ora mixture of these. Suitable emulsifying agents includenaturally-occurring gums, such as gum acacia and gum tragacanth;naturally occurring phosphatides, such as soybean lecithin, esters orpartial esters derived from fatty acids; hexitol anhydrides, such assorbitan monooleate; and condensation products of these partial esterswith ethylene oxide, such as polyoxyethylene sorbitan monooleate. Theemulsion may also contain sweetening and flavoring agents. Syrups andelixirs may be formulated with sweetening agents, such as glycerol,sorbitol or sucrose. Such formulations may also contain a demulcent, apreservative, a flavoring or a coloring agent.

Additionally, the pharmaceutical compositions of the invention may be inthe form of a sterile injectable preparation, such as a sterileinjectable aqueous emulsion or oleaginous suspension. This emulsion orsuspension may be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents which havebeen mentioned above. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxic parenterallyacceptable diluent or solvent, such as a solution in 1,2-propane-diol.The sterile injectable preparation may also be prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution, and isotonic sodium chloride solution. Inaddition, sterile fixed oils may be employed as a solvent or suspendingmedium. For this purpose any bland fixed oil may be employed includingsynthetic mono- or di-glycerides. In addition, fatty acids such as oleicacid may likewise be used in the preparation of injectables.

Generally, the compounds of the present invention useful in the methodsof the present invention are substantially insoluble in water and aresparingly soluble in most pharmaceutically acceptable protic solventsand in vegetable oils. However, the compounds are generally soluble inmedium-chain fatty acids (e.g., caprylic and capric acids) ortriglycerides and have high solubility in propylene glycol esters ofmedium-chain fatty acids. Also contemplated in the invention arecompounds which have been modified by substitutions or additions ofchemical or biochemical moieties which make them more suitable fordelivery (e.g., increase solubility, bioactivity, palatability, decreaseadverse reactions, etc.), for example by esterification, glycosylation,PEGylation, etc.

In a preferred embodiment, the compounds of the present invention may beformulated for oral administration in a lipid-based formulation suitablefor low solubility compounds. Lipid-based formulations can generallyenhance the oral bioavailability of such compounds. As such, a preferredpharmaceutical composition of the invention comprises a therapeuticallyor prophylactically effective amount of a compound of the presentinvention, together with at least one pharmaceutically acceptableexcipient selected from the group consisting of: medium chain fattyacids or propylene glycol esters thereof (e.g., propylene glycol estersof edible fatty acids such as caprylic and capric fatty acids) andpharmaceutically acceptable surfactants such as polyoxyl 40 hydrogenatedcastor oil.

In an alternative preferred embodiment, cyclodextrins may be added asaqueous solubility enhancers. Preferred cyclodextrins includehydroxypropyl, hydroxyethyl, glucosyl, maltosyl and maltotriosylderivatives of α-, β-, and γ-cyclodextrin. A particularly preferredcyclodextrin solubility enhancer is hydroxypropyl-β-cyclodextrin (HPBC),which may be added to any of the above-described compositions to furtherimprove the aqueous solubility characteristics of the compounds of thepresent invention. In one embodiment, the composition comprises 0.1% to20% hydroxypropyl-β-cyclodextrin, more preferably 1% to 15%hydroxypropyl-β-cyclodextrin, and even more preferably from 2.5% to 10%hydroxypropyl-β-cyclodextrin. The amount of solubility enhancer employedwill depend on the amount of the compound of the present invention inthe composition.

F. Combination Therapy

It is also possible to combine any compound of the present inventionwith one or more other active ingredients useful in the treatment of HCVinfection, including compounds, in a unitary dosage form, or in separatedosage forms intended for simultaneous or sequential administration to apatient in need of treatment. When administered sequentially, thecombination may be administered in two or more administrations. In analternative embodiment, it is possible to administer one or morecompounds of the present invention and one or more additional activeingredients by different routes.

The skilled artisan will recognize that a variety of active ingredientsmay be administered in combination with the compounds of the presentinvention that may act to augment or synergistically enhance the viralinhibiting activity of the compounds of the invention. Such activeingredients include anti-HCV agents. Anti-HCV agents include agents thattarget the virus as well as agents that have an immunomodulatory effect.For example, anti-HCV agents include, but are not limited to,interferon, including, for example without limitation, IFN-α, ribavirinor prodrugs or derivatives thereof; a glucosidase inhibitor, proteaseinhibitors, polymerase inhibitors, helicase inhibitors, a Toll-likereceptor agonist, a caspase inhibitor and a glycosidase inhibitor.Furthermore, the compounds of the invention may also be administered incombination with other compounds that affect IRES activity known to oneof skill in the art.

According to the methods of the invention, the combination of activeingredients may be: (1) co-formulated and administered or deliveredsimultaneously in a combined formulation; (2) delivered by alternationor in parallel as separate formulations; or (3) by any other combinationtherapy regimen known in the art. When delivered in alternation therapy,the methods of the invention may comprise administering or deliveringthe active ingredients sequentially, e.g., in separate solution,emulsion, suspension, tablets, pills or capsules, or by differentinjections in separate syringes. In general, during alternation therapy,an effective dosage of each active ingredient is administeredsequentially, i.e., serially, whereas in simultaneous therapy, effectivedosages of two or more active ingredients are administered together.Various sequences of intermittent combination therapy may also be used.

To assist in understanding the present invention, the following Examplesare included. The experiments relating to this invention should not, ofcourse, be construed as specifically limiting the invention and suchvariations of the invention, now known or later developed, which wouldbe within the purview of one skilled in the art are considered to fallwithin the scope of the invention as described herein and hereinafterclaimed.

Examples

The present invention is described in more detail with reference to thefollowing non-limiting examples, which are offered to more fullyillustrate the invention, but are not to be construed as limiting thescope thereof The examples illustrate the preparation of certaincompounds of the invention, and the testing of these compounds in vitroand/or in vivo. Those of skill in the art will understand that thetechniques described in these examples represent techniques described bythe inventors to function well in the practice of the invention, and assuch constitute preferred modes for the practice thereof However, itshould be appreciated that those of skill in the art should in light ofthe present disclosure, know that many changes can be made in thespecific methods that are disclosed and still obtain a like or similarresult without departing from the spirit and scope of the invention.

Example 1 Preparation of Compounds of the Invention Example 1APreparation of tert-Butyl3-amino-5-ethyl-4,6-dimethylthieno[2,3-B]pyridine-2-carboxylate(compound 3)

Step A: To a mixture of 2-cyanothioacetamide (6.33 g, 63.3 mmol) andtriethylamine (6.39 g, 8.81 mL, 63.3 mmol) in ethanol (100 mL) at roomtemperature was added 3-ethylpentane-2,4-dione (8.1 g, 8.50 mL, 63.3mmol). After stirring at 60° C. for 1 h, the mixture was cooled to roomtemperature and poured into cold water (800 mL). The precipitate,5-ethyl-2-mercapto-4,6-dimethylnicotinonitrile, was collected byfiltration, washed with hexanes (30 mL×2) and dried in air to give ayellow powder (10.87 g, 89%). LC/MS reveals a single component (MSES+m/z: 193) that was used without additional purification.

Step B: A mixture of 5-ethyl-2-mercapto-4,6-dimethylnicotinonitrile(3.84 g, 20.0 mmol) prepared above, tert-butyl bromoacetate (4.29 g,22.0 mmol) and K₂CO₃ powder (6.90 g, 50.0 mmol) in DMF (50 mL) wasstirred at room temperature for 20 min. and then at 80° C. for 24 h. Themixture was then poured into cold water (500 mL). The precipitate wascollected by filtration, washed with water and dried in air to give ayellow powder (5.90 g, 96%) to give the title compound, tert-butyl3-amino-5-ethyl-4,6-dimethylthieno[2,3-B]pyridine-2-carboxylate.

The following compounds were prepared in the same fashion as describedabove, by using various 1,3-diketones and 2-haloacetic acid derivatives,or 2-haloacetonitrile and 2-halomethylheteroaromatics: Compounds 2, 18,19, 20, 21, 22, 26, 28, 29, 30, and 56.

Example 1B Preparation of tert-butyl3-amino-4-thiophen-2-yl-6,7,8,9-tetrahydro-5H-1-thia-10-aza-cyclohepta[f]indene-2-carboxylate(compound 45)

Step A: To a solution of thiophene-2-carbaldehyde (20.0 g, 0.18 mol) and2-cyanothioacetamide (17.8 g, 0.18 mol) in methanol (400 mL), was addedneutral Al₂O₃ (106 g). The suspension was stirred at room temperaturefor 48 hrs, and then filtered. The solid was washed with methanol andthe filtrates were combined and the solvent was then removed byevaporation to afford 2-cyano-3-thien-2-ylprop-2-enethioamide (30 g,87%).

Step B: A mixture of 2-cyano-3-thien-2-ylprop-2-enethioamide (4.2 g, 21mmol), cycloheptone (2.4 g, 21 mmol) and piperidine (1.8 g, 22 mmol) inethanol (100 mL) was heated to 80° C. overnight. After removal of thesolvent, the residue was treated with water and extracted with ethylacetate. The organic phase was washed with brine and dried overanhydrous sodium sulfate. The crude product, obtained after removal ofthe solvent, was purified by flash chromatography to afford2-mercapto-4-thien-2-yl-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridine-3-carbonitrile(1.6 g, 25%).

Step C: A mixture of2-mercapto-4-thien-2-yl-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridine-3-carbonitrile(143 mg, 0.5 mmol), tent-butyl bromoacetate (97 mg, 0.5 mmol) andtriethyl amine (51 mg, 0.5 mmol) in ethanol (20 mL) was stirred at roomtemperature overnight, followed by the addition of NaOMe (30 mg, 0.56mmol). The mixture was then refluxed for 30 min., and poured onto ice.The precipitate is filtered and washed with water to furnish puret-butyl3-amino-4-thiophen-2-yl-6,7,8,9-tetrahydro-5H-1-thia-10-aza-cyclohepta[f]indene-2-carboxylate.

The following compounds were prepared in the same fashion as describedabove, using various aldehydes, ketones and 2-haloacetic acidderivatives: Compounds 31, 32, 33, 34, and 46.

Example 1C Preparation of t-butyl3-amino-4,6-dimethyl-5-(2-morpholin-4-yl-ethoxy)-thieno[2,3-b]pyridine-2-carboxylate(compound 67)

Step A: To a solution of pentane-2,4-dione (67.5 g, 67.5 mmol) intoluene (1.5 L) was added portionwise, Pb(OAc)₄ (300 g, 67.5 mmol) atroom temperature. The reaction mixture was then heated to 110° C. andstirred overnight. After cooling to room temperature, the reactionmixture was quenched by water (800 mL). The aqueous layer was extractedwith EtOAc (800 mL×3). The combined organic layers were washed withbrine, dried over anhydrous MgSO₄ and concentrated under reducedpressure to give crude 1-acetyl-2-oxo-propyloxy acetate (64 g, 60%).

Step B: To a solution of the above compound (40 g, 25.3 mmol) in ethanol(1 L) was added Et₃N (25.6 g, 25.3 mmol). After stirring for 0.5 h, asolution of 2-cyanothioacetamide (25.3 g, 25.3 mmol) in ethanol (150 mL)was added dropwise. The reaction mixture was stirred at room temperatureovernight. Then, the reaction was quenched by water (600 mL) and theaqueous layer was extracted with EtOAc (600 mL×3). The combined organiclayer was washed with brine, dried over anhydrous MgSO₄ and concentratedunder reduced pressure to give a crude product, which was dissolved withethanol (800 mL) and treated with Et₃N (17.3 g, 17.1 mmol) and stirringfor an additional 0.5 h. To this solution, a solution of t-butylchloroacetate (25.7 g, 17.2 mmol) in ethanol (150 mL) was addeddropwise, and then the reaction mixture was heated to reflux and stirredfor 3 h. The solvent was evaporated under reduced pressure, the residuewas treated with water (800 mL), and the precipitate was collected anddissolved in ethanol (500 mL). Sodium methoxide (2.4 g) was added to thesolution portionwise, the mixture was heated to reflux for 3 h and thenconcentrated. The residue that was obtained was then treated with water(800 mL), and the solid was collected via filtration to give tert-butyl5-acetoxy-3-amino-4,6-dimethyl-thieno[2,3-b]pyridine-2-carboxylate (19.6g, 78%).

Step C: To a solution of tert-butyl5-acetoxy-3-amino-4,6-dimethyl-thieno[2,3-b]pyridine-2-carboxylate (19.6g, 5.8 mmol) in methanol (300 mL), was added a 5% solution of aqueousLiOH (150mL) and the mixture was stirred overnight. The precipitate wascollected and washed with water to provide tert-butyl3-amino-5-hydroxy-4,6-dimethyl-thieno[2,3-b]pyridine-2-carboxylate (12g, 71%).

Step D: The tert-butyl3-amino-5-hydroxy-4,6-dimethyl-thieno[2,3-b]pyridine-2-carboxylateobtained above (12 g, 40 mmol) was dissolved in CH₃CN (300 mL), followedby the addition of K₂CO₃ (11 g, 80 mmol) and 1-bromo-2-chloroethane (5.7g, 40 mmol). The mixture was heated to reflux for 8 h, water (100 mL)was added and the mixture was extracted with ethyl acetate (60 mL×3).The organic layers were washed with 0.5% aqueous sodium hydroxide (150mL) and brine, dried over anhydrous Na₂SO₄ and then concentrated to givetert-butyl3-amino-5-(2-chloroethoxy)-4,6-dimethyl-thieno[2,3-b]pyridine-2-carboxylateas a yellow solid (6.9 g, 50%).

Step E: tert-Butyl3-amino-5-(2-chloroethoxy)-4,6-dimethyl-thieno[2,3-b]pyridine-2-carboxylate,prepared above (50 mg, 0.22 mmol), was dissolved in CH₃CN (5 mL),followed by the addition of K₂CO₃ (41 mg, 0.30 mmol), morpholine (26 mg,0.30 mmol) and a catalytic amount of K1. The mixture was heated toreflux overnight, the solvent was removed under reduced pressure, waterwas added and the residue was extracted with ethyl acetate (10 mL), andthe organic phases were washed with water (5 mL×3), brine, dried overanhydrous Na₂SO₄, concentrated and purified by preparative HPLC tofurnish the title compound, tert-butyl3-amino-4,6-dimethyl-5-(2-morpholin-4-yl-ethoxy)-thieno[2,3-b]pyridine-2-carboxylate(20 mg, 30%).

The following compounds were prepared in the same fashion as describedabove: Compounds 66, 68, 69, 70, 71, 72, 73, and 74.

Example 1D Preparation of tert-butyl3-amino-4,6-dimethyl-5-(pyrrolidine-1-carbonyloxy)-thieno[2,3-b]pyridine-2-carboxylate(compound 75)

tert-Butyl3-amino-5-hydroxy-4,6-dimethyl-thieno[2,3-b]pyridine-2-carboxylate,prepared in example 1C, Step C, (40 mg, 0.14 mmol) in CH₃CN (1 mL) wascombined with Et₃N (52 mg, 0.52 mmol) and pyrrolidine-1-carbonylchloride (51 mg, 0.39 mmol) at room temperature. After stirring for 3 h,the mixture was evaporated under reduced pressure, water was added andextracted with ethyl acetate (3 mL), washed with water (3 mL×3), brine,dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. Theresidue was purified by preparative HPLC to provide tert-butyl3-amino-4,6-dimethyl-5-(pyrrolidine-1-carbonyloxy)-thieno[2,3-b]pyridine-2-carboxylate(41 mg, 52%).

The following compound was prepared in the same fashion as describedabove: Compound 76.

Example 1E Preparation of5-ethyl-4,6-dimethyl-2-(5-phenyl-[1,3,4]oxadiazol-2-yl)-thieno[2,3-b]pyridin-3-ylamine (compound 6)

To a solution of3-amino-5-ethyl-4,6-dimethyl-thieno[2,3-b]pyridine-2-carboxylic acid(0.16 g, 0.63 mmol) in a mixture of dichloroethane (8 mL) and DMF (2 mL)was added PS-carbodiimide (1.0 g, 1.26 mmol). The mixture was shaken for15 min at room temperature, followed by the addition of5-phenyltetrazole (0.042 g, 0.32 mmol). Then, the mixture was shaken at80° C. overnight. The resin was then removed by filtration and washedwith hot chloroform (2×5 mL). The filtrate was evaporated in vacuum todryness and purified by chromatography (silica gel,dichloromethane/ethyl acetate, 9/1) to furnish the product5-ethyl-4,6-dimethyl-2-(5-phenyl-[1,3,4]oxadiazol-2-yl)-thieno[2,3-b]pyridin-3-ylamine (0.063 g, 56%).

The following compounds were prepared in the same fashion as describedabove: Compounds 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 23.

Example 1F Preparation of4,5,6-trimethyl-2-(3-phenyl-[1,2,4]oxadiazol-5-yl)-thieno[2,3-b]pyridin-3-ylamine(compound 24)

To a solution of N-hydroxy-benzamidine (41 mg, 0.30 mmol) in DCM (10 mL)was added PS-DIEA (240 mg, 0.90 mmol), followed by the addition ofchloroacetyl chloride (0.36 mL, 0.45 mmol). The mixture was shaken atroom temperature for 24 hr and filtered to remove the resin. The solventwas then replaced with toluene and the mixture obtained was stirred at120° C. in a sealed tube overnight. The toluene was then replaced withDMF (10 mL) and to the solution was added K₂CO₃ (124 mg, 0.90 mmol) and2-mercapto-4,5,6-trimethyl-nicotinonitrile (54 mg, 0.30 mmol). Themixture was stirred at 80° C. overnight before pouring it into water.The precipitate was collected and purified via flash chromatography toprovide the title product,4,5,6-trimethyl-2-(3-phenyl-[1,2,4]oxadiazol-5-yl)-thieno[2,3-b]pyridin-3-ylamine (21 mg, 21%).

The following compound was prepared in similar fashion as describedabove: Compound 25.

Example 1G Preparation of cyclopentanecarboxylic acid(3-cyano-5-ethyl-4,6-dimethyl-thieno[2,3-b]pyridin-2-yl)amide (compound49)

Step A: To a mixture of methyl3-amino-5-ethyl-4,6-dimethylthieno[2,3-b]pyridine-2-carboxylate (7.92 g,30.0 mmol), prepared analogously to the procedure described in example1A, and CuI (11.40 g, 60.0 mmol) in acetonitrile (150 mL), was addedn-butyl nitrite (6.18 g, 7.12 mL, 60.0 mmol). The mixture was stirred at50° C. for 24 h, and then poured into water (500 mL). To this mixturewas added dichloromethane (100 mL), followed by the addition ofconcentrated ammonium hydroxide dropwise with stirring, until all theprecipitate had dissolved. The dichloromethane layer was separated andwashed with water, brine and dried over anhydrous Na₂SO₄. The crudeproduct was obtained after the removal of the solvent andchromatographed (CH₂Cl₂/hexanes, 1/9) to give methyl3-iodo-5-ethyl-4,6-dimethylthieno[2,3-b]pyridine-2-carboxylate (5.18 g,46%). MS (ES⁺) m/z: 376.

Step B: To the solution of methyl3-iodo-5-ethyl-4,6-dimethylthieno[2,3-b]pyridine-2-carboxylate preparedabove (5.18 g, 13.8 mmol) in NMP (100 mL), was added CuCN (2.47 g, 27.6mmol). The mixture was stirred at room temperature overnight. Work-upwas conducted as above and the crude product was chromatographed(EtOAc/CH₂Cl₂, 2.5/97.5) to give pure methyl3-cyano-5-ethyl-4,6-dimethylthieno[2,3-b]pyridine-2-carboxylate (2.70 g,71%). MS (ES+) m/z: 275.

Step C: To a solution of methyl3-cyano-5-ethyl-4,6-dimethylthieno[2,3-b]pyridine-2-carboxylate (2.70 g,9.9 mmol), prepared above, in ethanol (100 mL), was added aqueous NaOH(1.25 N, 15.7 mL, 19.6 mmol), and the mixture was stirred for 2 h at 60°C. The volatiles were removed under reduced pressure and the residue wasdissolved in water (150 mL) and then acidified using concentrated HCluntil the pH was adjusted to 5-6. The precipitate was collected viafiltration and washed thoroughly with water, dried in air to give3-cyano-5-ethyl-4,6-dimethylthieno[2,3-b]pyridine-2-carboxylic acid(2.38 g, 93%). MS (ES) m/z: 259.

Step D: 3-Cyano-5-ethyl-4,6-dimethylthieno[2,3-b]pyridine-2-carboxylicacid (1.00 g, 3.85 mmol) was mixed with triethylamine (0.78 g, 1.07 mL,7.69 mmol), diphenylphosphoryl azide (2.12 g, 7.69 mmol) and anhydroust-butanol (15 mL). The mixture was stirred at 80° C. for 8 h, followedby the removal of the volatiles under vacuum. To the residue was addeddichloromethane (150 mL) and saturated NaHCO₃ (40 mL), and the mixturewas filtered after stirring at room temperature for 0.5 h. The organicswere separated from the filtrate and dried over anhydrous Na₂SO₄. Afterremoval of the solvent, the residue was chromatographed to give(3-cyano-5-ethyl-4,6-dimethyl-thieno[2,3-b]pyridin-2-yl)-carbamic acidtert-butyl ester. MS (ES⁺) m/z: 332.

Step E: The t-butylcarbamate obtained above was treated with TFA (20 mL)at room temperature for 3 h, and then evaporated to dryness undervacuum. The residue was stirred in aqueous K₂CO₃ (50 mL) for 2 h. Theprecipitate was collected by filtration, washed and dried in air tofurnish essentially pure product,2-amino-5-ethyl-4,6-dimethylthieno[2,3-b]pyridine-3-carbonitrile,compound 35, (0.59 g, 66% from the carboxylic acid in step C). MS (ES⁺)m/z: 232.

Step F: To a solution of2-amino-5-ethyl-4,6-dimethylthieno[2,3-b]pyridine-3-carbonitrile (23 mg,0.10 mmol) in pyridine (2.5 mL) was added cyclopentanecarbonyl chloride(15 mg, 0.11 mmol). The mixture was stirred at room temperatureovernight, poured into water (10 mL) and extracted with DCM (10 mL). Theorganic layer was separated and washed with HCl (2 N, 2×3 mL), water(3×3 mL) and brine (3 mL), dried over anhydrous Na₂SO₄. The solvent wasremoved under reduced pressure to provide essentially purecyclopentanecarboxylic acid(3-cyano-5-ethyl-4,6-dimethyl-thieno[2,3-b]pyridin-2-yl)amide (28 mg,86%).

The following compounds were prepared in the same fashion as describedabove: Compounds 50 and 53.

Example 1H Preparation of2-(4-methoxyphenyl)-4-thiophen-2-yl-6,7,8,9-tetrahydro-5H-1-thia-10-aza-cyclohepta[f]indene-3-carbonitrile(compound 63)

Step A: tert-Butyl nitrite (1.66 mL, 0.0138 mol) and CuBr₂ (2.5 g, 0.011mol) were heated in acetonitrile (80 mL) to reflux.2-Amino-4-thiophen-2-yl-6,7,8,9-tetrahydro-5H-1-thia-10-aza-cyclohepta[f]indene-3-carbonitrile(3.0 g, 0.0092 mol) in 10 mL of acetonitrile was then added to themixture. The mixture was refluxed for 80 minutes, after which thesolvent was removed and the residue was partitioned between 500 mL ofEtOAc and 100 mL of aqueous ammonia. The organic layer was separated,washed with brine and dried over MgSO₄. Then, the residue was purifiedby chromatography to provide ca.1 g of2-bromo-4-thiophen-2-yl-6,7,8,9-tetrahydro-5H-1-thia-10-aza-cyclohepta[f]indene-3-carbonitrile.

Step B: To a Schlenk tube was added2-bromo-4-thiophen-2-yl-6,7,8,9-tetrahydro-5H-1-thia-10-aza-cyclohepta[f]indene-3-carbonitrile(45 mg, 0.116 mmol), p-methoxyphenyl boronic acid (21 mg, 0.138 mmol),NaHCO₃ (29 mg, 0.348 mmol), Bu₄NBr (8 mg, 0.023 mmol) and 1 mL ofDME/H₂O (4/1). After evacuating and back-filling with nitrogen twice, acatalytic amount of Pd(PPh₃)₄ was added. The sealed mixture was heatedto 80° C. overnight. After pouring into water, the mixture was extractedwith ethyl acetate. The organic phase was washed with brine, dried overanhydrous MgSO₄ and concentrated. The residue was purified by flashchromatography to provide 30 mg of the title compound,2-(4-methoxyphenyl)-4-thiophen-2-yl-6,7,8,9-tetrahydro-5H-1-thia-10-aza-cyclohepta[f]indene-3-carbonitrile.

The following compounds were prepared in the same fashion as describedabove: Compounds 61 and 62.

Example 11 Preparation of t-butyl5-ethyl-3-(4-methoxy-phenyl)-4,6-dimethyl-thieno[2,3-b]pyridine-2-carboxylate(compound 37)

To a solution of compound tert-butyl5-ethyl-3-iodo-4,6-dimethyl-thieno[2,3-b]pyridine-2-carboxylate (42 mg,0.1 mmol) in 0.5 mL of DME/H₂O (1/1) was added K₂CO₃ (41 mg, 0.3 mmol),Pd(PPh₃)₄ (12 mg, 0.01 mmol), p-methoxyphenyl boronic acid (18 mg, 0.12mmol). The mixture was then heated to reflux for 16 h. After cooling toroom temperature, the solvent was evaporated under reduced pressure. Theresidue was dissolved with ethyl acetate (2 mL), washed with water (2mL×3), brine, dried over anhydrous Na₂SO₄ and evaporated under reducedpressure. The crude solid was purified by preparative HPLC to providet-butyl5-ethyl-3-(4-methoxy-phenyl)-4,6-dimethyl-thieno[2,3-b]pyridine-2-carboxylate(12 mg, 30%).

The following compounds were prepared in the same fashion as describedabove: Compounds 36, 37, 39, 40, 42, and 43.

Example 1J Preparation ofN-(3-cyano-4-thiophen-2-yl-6,7,8,9-tetrahydro-5H-1-thia-10-aza-cyclohepta[f]inden-2-yl)-3-trifluoromethyl-benzamide(compound 58)

A mixture of2-amino-4-thiophen-2-yl-6,7,8,9-tetrahydro-5H-1-thia-10-aza-cyclohepta[f]indene-3-carbonitrile,prepared using the chemistry described in Example 1G (100 mg, 0.31mmol), 3-trifluoromethyl-benzoyl chloride (77 mg, 0.37 mmol) and acatalytic amount of DMAP in pyridine (20 mL) was heated under reflux forone day. After concentration, the residue was partitioned between EtOAcand H₂O. The organic layer was washed with brine, dried over MgSO₄ andconcentrated. The residue was purified by chromatography to giveN-(3-cyano-4-thiophen-2-yl-6,7,8,9-tetrahydro-5H-1-thia-10-aza-cyclohepta[f]inden-2-yl)-3-trifluoromethyl-benzamide(40 mg, 26%).

The following compound was prepared in the same fashion as describedabove: Compound 57.

Example 1K Preparation of4-hydroxymethyl-2-methyl-6-phenyl-6,7-dihydro-5H-9-thia-1,5,7-triaza-fluoren-8-one(compound 38)

Step A: To a solution of ethyl acetoacetate (60 g, 0.38 mol) in ethanol(1.2 L) was added Et₃N (38.5 g, 0.38 mol). After stirring for 0.5 h, asolution of cyanothioacetamide (38.0 g, 0.38 mol) in ethanol (200 mL)was added dropwise. The reaction mixture was stirred at room temperatureovernight after which it was quenched with water (750 mL). The aqueouslayer was extracted with ethyl acetate (750 mL×3). The combined organiclayers were washed with brine, dried over anhydrous MgSO₄ andconcentrated under reduced pressure to give a crude product (55 g, 65%),which was dissolved in 800 mL of ethanol. Triethyl amine (25.3 g, 0.25mol) was added and after stirring for 0.5 h, a solution of2-chloroacetamide (23.3 g, 0.25 mol) in ethanol (250 mL) was addeddropwise. After the addition, the reaction mixture was heated to refluxand stirred for 3 h. After cooling to room temperature, most of thesolvent was evaporated under reduced pressure. To the approximately 50mL of remaining solution, 900 mL of water was added, and afterfiltration, the crude solid was collected (68%, 47 g). The solid wasdissolved into 600 mL of ethanol. Solid sodium methoxide (3.0 g) wasadded portionwise, and the mixture was heated to reflux for 3 h. Aftercooling to room temperature, the reaction was concentrated under reducedpressure. Water (800) mL was added to the residue, and after filtrationethyl 3-amino-2-carbamoyl-6-methyl-thieno[2,3-b]pyridine-4-carboxylatewas collected (85%, 40 g).

Step B: To a suspension of LiAlH₄ (22 g, 0.57 mol) in anhydrous THF (250mL) was added a solution of ethyl3-amino-2-carbamoyl-6-methyl-thieno[2,3-b]pyridine-4-carboxylate (40 g,143 mmol) in anhydrous THF (250 mL) at −40° C. dropwise. The reactionwas then allowed to warm to room temperature and the mixture was heatedto reflux and stirred for 5 h. After cooling to room temperature, thereaction was quenched by aqueous NaOH (2N, 22 mL) at 0° C., filteredthrough Celite and washed with THF (50 mL×5). The filtrate was collectedand evaporated under reduced pressure to give crude3-amino-4-hydroxymethyl-6-methyl-thieno[2,3-b]pyridine-2-carboxamide(74%, 25 g).

Step C: To a solution of3-amino-4-hydroxymethyl-6-methyl-thieno[2,3-b]pyridine-2-carboxamide(100 mg, 0.42 mmol) in acetic acid (2 mL) was added benzaldehyde (53 mg,0.50 mmol) and the mixture was heated to reflux for 16 h. After coolingto room temperature, water (2 mL) and ethyl acetate (2 mL) were added toit. The reaction was further extracted with ethyl acetate (2 mL×3). Thecombined organic layers were washed with brine, dried over Na₂SO₄ andevaporated under reduced pressure. The residue was purified bypreparative HPLC to give the title compound,4-hydroxymethyl-2-methyl-6-phenyl-6,7-dihydro-5H-9-thia-1,5,7-triaza-fluoren-8-one (16 mg, 12%).

Example 2 Screening of Low Molecular Weight Compounds Using a Cell-BasedHCV IRES Monocistronic Translation Assay

Chemical libraries are screened using a cell-based monocistronic HCVIRES-regulated translation assay designed to closely mimic natural HCVmRNA translation and then compound analogs are made based on hits in thechemical libraries and screened as well. A DNA construct is prepared,termed pHCVIRESmono, in which HCV IRES sequences (HCV 2b, nucleotides18-347) are inserted between a promoter and the firefly luciferase(Fluc) reporter gene. A stably transfected HepG 2 (hepatoblastoma) cellline (termed HepGmono-4) or a Huh cell line (termed Huhmono 7), or aHela-cell line (termed Helamono), are established by transfection withthe pHCVIRESmono DNA by selecting for resistance to hygromycin.

Example 3 Determination of Selectivity for HCV IRES-RegulatedTranslation Using the Cell-Based Cap-Dependent Translation Assays

Since translation assays are used to screen HCV IRES inhibitors, theselected hits may specifically act on HCV IRES-driven translation or maymodulate general protein synthesis in mammalian cells. The compoundsthat act on general translation will most likely have significanttoxicity. To address this possibility, various cell-based cap-dependenttranslation assays are established for the further evaluation of allselected compounds. Plasmid DNAs containing 130 nucleotides of vectorsequence 5′ to Fluc are constructed. This construct is referred toherein as pLuc. A stable cell line is established in cap-dependenttranslation assays using 293T cells (a human embryonic kidney cellline). HepGmono-4 and pLuc are treated with compound for 20 hours andactivity is determined by quantifying the Fluc signal. A five-foldselectivity between the HCV IRES and cap-dependent translation isconsidered to be desirable. For example, using these cell-basedcap-dependent translation assays, Applicants identified compounds thatshowed IC₅₀ values that were at least 5-fold greater in thecap-dependent translation assays than in the HCV IRES translation assay.

Additionally, western blotting assays are used to further demonstratethat the compounds selectively inhibit HCV IRES-driven translation. BothHepGmono-4 and pLuc cells are treated with the compounds as describedabove, following treatment with the test compounds for 20 hours, cellsare collected and lysed in Laminin buffer containing 0.5% SDS. Proteinsare separated on a 10% SDS-PAGE, then transferred onto a nitrocellulosemembrane, and blotted using antibodies against Fluc (RDI) and β-actin(Oncogene). For example, some of the compounds of the present inventionwere tested in this manner and as expected, the compounds thatselectively inhibited HCV IRES-driven translation in assays using Flucsignal as an end point showed comparable reductions of the luciferasereporter protein levels in HepGmono-4 cells and were relatively inactiveagainst pLuc in the Western blot (data not shown). Importantly, thesecompounds did not inhibit the expression of endogenous 13-actin, thetranslation of which is cap-dependent in both cell lines. Consistently,compounds that did not show selectivity in the translation assaysinhibited protein accumulation in both the HCV IRES and cap-dependenttranslation assays (data not shown). As expected, the general proteintranslation inhibitor puromycin also inhibited both the HCV IRES-drivenand cap-dependent protein production (data not shown). Therefore, theWestern blot results confirm that the compounds of the present inventionselectively inhibit HCV IRES-driven translation.

Testing conditions for these cell lines are optimized and the effects ofmRNA level on activity of the compounds are controlled by quantifyingFluc mRNA levels by RT real-time PCR. For example, some of the compoundsof the present invention were tested in this manner, and no significantdifferences in Fluc mRNA levels were observed between the HepGmono-4, orthe Hela cells, or the Huh cells, and cap-dependent translation celllines used (data not shown).

Example 4 Evaluation of the Selectivity for HCV IRES-Driven TranslationUsing Cellular IRES-Mediated Translation Assays

A number of human mRNAs have been shown to harbor IRES elements (18, 19,39, 44, 45, 91, 126, 130). Although the primary sequences and secondarystructures of the HCV IRES are different from those of cellular IRESs,an important test for selectivity is to determine whether the selectedcompounds are active against cellular IRESs. The VEGF IRES has poorinitiation activity in in vitro assays, but demonstrates substantialactivity in cell-based translation assays (18, 45). For example, some ofthe compounds of the present invention were tested and all of thecompounds that had good selectivity with respect to cap-dependenttranslation exhibited at least 5-fold higher IC₅₀ values against theVEGF IRES than against the HCV IRES (data not shown). These dataindicate that the selected compounds have selectivity against viralIRESs. In addition to having different structures, the VEGF IRES alsohave different interactions with non-canonical cellular translationfactors. These differences may contribute to the selectivity of the HCVIRES inhibitors that we have identified. Cellular IRESs appear tofunction under conditions, such as stress or hypoxia, when cap-dependenttranslation is blocked (19, 126). Therefore, the lack of selectivitywith respect to cellular IRESs may not necessarily be predictive ofclinical toxicity.

Example 5 Evaluation of Cytotoxicity

Effects on cell proliferation are a critical issue for any drugdiscovery effort. Therefore, a cell proliferation/cytotoxicity assay isused to eliminate any compounds that affect mammalian cell growth. Theeffects of the selected hits on cell proliferation are tested in humancell lines 293 T and Huh? (a human hepatoblastoma cell line). Cells aregrown in Dulbecco's modified Eagle's medium supplemented with 10% fetalbovine serum, L-glutamine, penicillin, and streptomycin. Cells in logphase are treated with test compounds for three days, with 250 μM beingthe highest concentration of test compound used. The effect of thecompounds on cell proliferation is assessed by using the CellTiter 96AQueous One Solution Cell Proliferation Assay (Promega, Madison, Wis.).Compounds that have at least 5-fold higher CC₅₀ values relative to IC₅₀values in HepGmono-4 are considered to have a sufficient window betweenactivity and cytotoxicity and, hence, are selected for furtherevaluation. For example, some of the compounds of the present inventionwere tested in this manner, and importantly, all compounds that had goodselectivity with respect to cap-dependent translation also demonstrateda greater than 5-fold ratio of CC₅₀ to IC₅₀ values.

Example 6 Evaluation of the Efficacy of the Compounds in the HCVReplicon System

The lack of reliable and readily accessible cell-culture and smallanimal models permissive for HCV replication has limited the developmentof new anti-HCV agents. Self-replicating subgenomic HCV systems, termedHCV replicons, have recently been described and have been widely used toassess the efficacy of anti-HCV inhibitors (8, 70, 104). Interferon(IFN) α and inhibitors of the HCV protease and polymerase have beenreported to be active in the HCV replicon system (8, 17, 32, 68, 69,117).

HCV replicons that include bicistronic and monocistronic systems areidentified and assays for testing the HCV IRES inhibitors areestablished. In the bicistronic replicons, the HCV IRES directs theexpression of the selective marker (Neo and/or a Flue reporter), and theEMCV IRES mediates the expression of viral non-structural proteins. Inthe monocistronic replicon, the HCV IRES directly mediates viral proteinsynthesis. The HCV IRES inhibitors are analyzed in the bicistronicreplicon by quantifying the Fluc reporter signal. Replicon-containingcells are cultured with the compounds of the invention for 2 days.Interferon (IFN) α is used as a positive control. For example, some ofthe compounds of the present invention were tested in this manner, andthe experiments showed that compounds that selectively inhibited HCVIRES-mediated translation inhibited Fluc expression in the bicistronicreplicon in the micromolar range.

In the following table (Table 1),

*=replicon or HCV-PV IC50>2 uM

**=replicon or HCV-PV IC50 between 0.5 uM and 2 uM

***=replicon or HCV-PV IC50<0.5 uM

Replicon IC50 values are determined by firefly luciferase signal.

HCV-PV IC50 values are determined by viral RNA reduction.

TABLE 1 Replicon Mass Compound IC50 s HCV-PV Melting Spec Number μM IC50uM Point (° C.) [M + H] NMR Data 1 * 203-204 2 * 318.0 3 ** 135-136307.18 ¹H NMR (CDCl₃, 300 MHz) δ (ppm) 1.16 (t, 3H), 1.59 (s, 9H), 2.64(s, 3H), 2.72 (s, 3H), 2.74-2.80 (q, 2H), 6.13 (s, b, 2H) 4 * 5 * **413.2 6 ** 306-307 351.29 ¹H NMR (CDCl₃, 300 MHz) δ (ppm) 1.17 (t, 3H),2.68 (s, 3H), 2.75-2.87 (m, 5H), 6.25 (s, br, 2H), 7.49-7.62 (m, 3H),8.08-8.20 (m, 2H) 7 ** 268-270 351.29 8 ** 300-302 355.24 9 ** 284-286377.18 10 ** 320-322 355.24 11 * 302-303 371.25 12 * 331-332 371.25 13*** 261-262 351.29 14 *** 287-288 355.24 15 ** 284-286 371.25 16 ***306-308 338.27 17 ** 169-170 279.1 18 ** 197-200 435.23 19 *** 281-282343.25 20 ** 244-245 367.30 21 *** 248-250 337.28 22 *** 260-265 343.2323 ** 238-240 367.30 24 *** 276-278 337.25 ¹H NMR (CD₃COCD₃, 300 MHz) δ(ppm) 2.36 (s, 3H), 2.60 (s, 3H), 2.85 (s, 3H), 7.02 (s, br, 2H),7.55-7.65 (m, 3H), 8.13-8.21 (m, 2H) 25 ** 278-280 338.27 26 ** 284-285336.15 27 * 145-147 299.18 28 * 338.14 29 * ** 406.22 30 * 272.18 31 *430.92 32 * 370.89 33 * 356.88 34 * ** 386.87 35 * *** 223-225 232.23 ¹HNMR (CDCl₃, 300 MHz) δ (ppm) 1.11-1.16 (t, 3H), 2.56 (s, 3H), 2.67 (s,3H), 2.67-2.74 (q, 2H), 5.16 (s, 2H) 36 * 392.99 37 * 397.99 38 * 326.139 * 151-152 411.96 40 *   111-111.5 357.90 41 * 409.31 42 * 117-118401.91 43 * 152.1-152.6 401.92 44 * 431.36 45 * 202.9-203.1 401.00 46 *157.6-157.7 329.30 47 * 115-118 427.29 48 * 184-185 400.45 49 * 228-230328.26 ¹H NMR (CDCl₃, 300 MHz) δ (ppm) 1.17 (t, 3H), 1.61-2.13 (m, 8H),2.64 (s, 3H), 2.69-2.99 (m, 6H), 8.52 (s, 1H) 50 * 164-166 316.30 51 *52 * 53 * 189-190 302.24 54 * 219-220 55 * 212-3  354.98 56 *206.3-203-8 323.02 CDCl₃, 300 MHz, δ = 6.61 (b, 2H), 2.75 (q, J = 7.5Hz, 2H), 2.71 (s, 3H), 2.64 (s, 3H), 1.59 (s, 9H), 1.15 (t, J = 7.5 Hz,3H) 57 * 215.3-215.7 430.24 58 * 226.9-227.1 498.25 59 * 183-185 331.2460 * 196.4-196.7 389.26 61 * 159-162 412.36 62 * 210-211 431.34 63 *179.2-179.6 417.34 64 * 432.28 65 ** 109.6-110.2 307.8 CDCl₃, 300 MHz, δ= 7.93 (s, 1H), 2.78 (q, J = 7.8 Hz, 2H), 2.67 (s, 3H), 2.57 (s, 3H),1.61 (s, 9H), 1.17 (t, J = 7.8 Hz, 3H) 66 * 79-81 465.0 CDCl₃, 300 MHz,δ = 4.09 (s, 2H), 3.97 (s, 4H), 3.47-3.33 (m, 12H) 2.66 (s, 3H), 2.61(s, 3H), 2.43 (b, 3H), 1.57 (s, 9H) 67 * 102-104 408.0 CD₄O, 300 MHz, δ= 4.23 (t, J = 5.0 Hz, 2H), 4.15-3.87 (m, 4H), 3.72 (t, J = 5.1 Hz, 2H),3.460-3.40 (m, 4H), 2.73 (s, 3H), 2.58 (s, 3H), 1.56 (s, 9H) 68 ** 435.1CDCl₃, 300 MHz, δ = 4.17 (s, 2H), 4.06 (s, 2H), 3.86-3.78 (m, 4H)3.58-3.56 (m, 4H), 2.94 (s, 3H), 2.66 (s, 3H), 2.56 (b, 5H), 1.57 (s,9H) 69 ** 492.1 CD₄O, 300 MHz, δ = 4.16 (t, J = 4.5 Hz, 2H), 3.52 (b,6H), 3.35 (b, 4H), 3.29 (t, J = 1.5 Hz, 2H), 3.22 (t, J = 7.8 Hz, 2H),3.04 (t, J = 7.2 Hz, 2H), 2.91 (s, 6H), 2.74 (s, 3H), 2.58 (s, 3H), 2.13(b, 2H), 1.56 (s, 9H) 70 ** 406.0 CD₄O, 300 MHz, δ = 4.19 (t, J = 5.1Hz, 2H), 3.74 (d, J = 12.3 Hz, 2H), 3.64 (t, J = 4.8 Hz, 2H), 3.18-3.08(m, 2H), 2.73 (s, 3H), 2.57 (s, 3H), 2.02-1.86 (m, 6H), 1.56 (s, 9H)71 * 155.4-155.5 421.0 CD₄O, 300 MHz, δ = 4.00 (t, J = 4.8 Hz, 2H),3.34-3.29 (m, 8H), 3.02 (t, J = 5.1 Hz, 2H), 2.91 (s, 3H), 2.76 (s, 3H),2.58 (s, 3H), 1.57 (s, 9H) 72 * 365.9 CD₄O, 300 MHz, δ = 4.18 (t, J =4.5 Hz, 2H), 3.68 (t, J = 5.4 Hz, 2H), 3.01 (s, 6H), 2.76 (s, 3H), 2.60(s, 3H), 1.57 (s, 9H) 73 ** 423.0 CDCl₃, 300 MHz, δ = 6.06 (s, 2H), 3.86(t, J = 4.8 Hz, 2H), 2.86 (t, J = 4.5 Hz, 2H), 2.69 (s, 3H), 2.65-2.57(m, 5H), 2.49-2.46 (m, 2H), 2.37 (s, 3H), 2.28 (s, 6H), 1.57 (s, 9H)74 * 435.0 CDCl₃, 300 MHz, δ = 4.16 (t, J = 4.5 Hz, 2H), 3.76 (b, 4H),3.65 (b, 4H), 3.46 (t, J = 4.8 Hz, 2H) 3.21 (q, J = 6.9 Hz, 2H), 2.70(s, 3H), 2.60 (s, 3H), 1.57 (s, 9H) 1.41 (t, J = 7.2 Hz, 3H) 75 *decomposed 391.9 (M − H) (CDCl3, 300 MHz), δ 3.66 (t, J = 6.3 Hz, 2H),3.51 (t, J = 6.3 Hz, 2H), 2.61 (s, 3H), 2.54 (s, 3H), 2.06-1.95 (m, 6H),1.58 (s, 9H) 76 * decomposed 407.9 (CDCl3, 300 MHz), δ 3.78 (b, 8H),3.60 (b, 2H), 2.57 (s, 3H), 2.49 (s, 3H), 1.57 (s, 9H)

Example 7 Evaluation of the Activity of Compounds Using anHCV-Poliovirus Chimera

In an HCV-poliovirus (HCV-PV) chimera, the PV 5′ UTR is replaced by theHCV 5′ UTR and partial (the first 123 amino acids) core coding sequences(nucleotides 18 to 710 of HCV 1b) as shown in FIG. 1 (140). As aconsequence, the expression of poliovirus proteins is under regulationof the HCV IRES. Poliovirus is a picornavirus in which proteintranslation initiation is mediated by an IRES element located in the 5′UTR. At the 5′ end of the HCV-PV chimeric genome, there is thecloverleaf-like RNA structure of PV, an essential cis-acting replicationsignal ending with the genome-linked protein VPg. Replication kineticsof the HCV-PV chimera matches that of the parental poliovirus (Mahoney)and can result in cytopathic effects (CPE) in cell culture. Heptazyme, aribozyme that targets the HCV IRES, was shown to be active against thechimeric virus in cell culture (76, 77).

To evaluate compounds for activity against the chimeric virus, HeLacells are seeded and incubated at 37° C. under 5% CO₂ for 24 hours. Thecells are then infected with HCV-PV at a multiplicity of infection (MOI)at 0.1 for 30 min and then treated with compound for 1 day. The activityof compounds is determined by a change in cytopathic effect, plaqueassay, and/or viral RNA production (see e.g., Table 1) is thenquantified by RT real-time PCR using the HCV IRES primers and probes.

Example 8 Evaluation of the Activity of Compounds Against a Wild-TypePoliovirus (WT-PV) and the Poliovirus IRES Translation Assay (WT-PV MonoLuc)

A DNA construct is prepared, termed pPVIRESmono, in which PV IRESsequences are inserted (nucleotide number 1-742) between a promoter andthe firefly luciferase (Fluc) reporter gene. A stably transfected 293 Tcell line, is established by transfection with the pPVIRESmono DNA byselecting for resistance to hygromycin. As previously described, cellsare treated with compounds for 20 hours, and activity is determined byquantifying the Fluc signal. Table 2 provides data obtained using someof the compounds of the present invention. Additionally, to evaluateactivity of compounds against wild-type poliovirus, HeLa cells areseeded and incubated at 37° C. under 5% CO₂ for 24 hours. Cells are theninfected with wild-type poliovirus at a MOI at 0.1 for 30 minutes, andthen treated with compound for one day. The activity of compounds isdetermined by changes in cytopathic effect (see e.g., Table 2), plaqueassay, and RNA production determined by RT real time PCR usingpoliovirus IRES primers and probes.

Furthermore, if compounds are active in the poliovirus IRES or othervirus IRESs, then the compounds may also be useful for treating viralinfection by other viruses containing an IRES.

TABLE 2 Compound WTPV CPE WTPV CPE WTPV CPE Number (100 μM) (11.1 μM)(1.2 μM) 5 2 1 0 29 2 1 0 35 2 1 0 50 3 1 0 *A “1” in the WT-PV CPEcolumns indicates that the CPE (cytopathic effect) is decreased by25-50%. A “2” in the WT-PV CPE columns indicates that the CPE isdecreased by 50-75%. A “3” in the WT-PV CPE columns indicates that theCPE is decreased by 75-100%.

Example 9 In Vitro Translation Assay

In vitro translation assays can be used to distinguish between thecompounds that act on HCV IRES RNA or cellular translation factors. Inexemplary assays, the mRNA that will direct translation is a transcribedrunoff product from the T7 RNA polymerase promoter of the pHCVIRESmonoplasmid DNA generated with Ambion RNA MegaTranscript kit (Ambion, Inc.,Austin, Tex.). In vitro translation is performed using HeLa cell lysatesusing methods known to one of skill in the art. Preliminary resultsindicate that at least one of the compounds of the present invention hassignificantly higher activity against HCV IRES regulated translationafter preincubating the compound with the HCV IRES RNA transcripts thanafter preincubating with HeLa cell lysate for 30 min at 37° C. orwithout preincubation (data not shown). This suggests that this compoundmay interact with the HCV IRES RNA in the in vitro translation assay. Todemonstrate whether the compounds selectively act on the HCV IRES, pLucis used together with cellular IRES mRNA transcripts as controls for invitro translation.

All publications and patent applications cited herein are incorporatedby reference to the same extent as if each individual publication orpatent application was specifically and individually indicated to beincorporated by reference.

Although certain embodiments have been described in detail above, thosehaving ordinary skill in the art will clearly understand that manymodifications are possible in the embodiments without departing from theteachings thereof. All such modifications are intended to be encompassedwithin the claims of the invention.

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1-34. (canceled)
 35. A compound having the following formula:

wherein: X is: hydrogen; a cyano group; an amino group; a

a 5- or 6-membered heteroaryl; a C₆ to C₈ aryl, optionally substitutedwith: an alkoxy group, a cyano group, or a halogen; Y is: a halogen; anamino group; a

a —SO₂R_(x), where R_(x) is methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl or cyclohexyl; a—COOR_(x) group, where R_(x) is n-butyl, isobutyl, sec-butyl,tert-butyl, n-pentyl, n-hexyl, or cyclohexyl; a C₆ aryl, optionallysubstituted with: an alkoxy, or a cyano group; a —COR_(a) group, whereR_(a) is: an amino substituted with one or two C₁ to C₆ alkyls, wherethe alkyls are optionally substituted with a C₆ aryl; a —NHR_(b) groupwhere R_(b) is: a C₆ aryl optionally substituted with: a haloalkyl, ahalogen, or a haloalkoxy; a 5- or 6-membered heterocycle optionallysubstituted with a C_(i) to C₆ alkyl; a C₁ to C₆ alkyl; or a —SR_(x)group, where R_(x) is methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl or cyclohexyl; a 5 or6 membered heteroaryl substituted with: a C₆ aryl optionally substitutedwith: an alkoxy, a halogen, or a C₁ to C₆ alkyl; or a 5- or 6-memberedheteroaryl optionally substituted with: an alkoxy, a halogen, or a C₁ toC₆ alkyl; a C₁ to C₆ alkyl, optionally substituted with a —OR_(c), whereR_(c) is a C₆ aryl optionally substituted with one or more halogens; anitro group; a —NHR_(d) group, where R_(d) is a C₆ aryl optionallysubstituted with an alkoxy; a —NHCOR_(e) group where R_(e) is: a C₆ aryloptionally substituted with a haloalkyl, or a methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl,cyclopentyl or cyclohexyl; R is: a hydrogen; a haloalkyl; a methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl,n-pentyl, n-hexyl or cyclohexyl optionally substituted with hydroxyl; orR together with R₁ forms:

R₁ is: a C₆ aryl; a methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl or cyclohexyl; aOCOR_(f) where R_(f) is a 5- or 6-membered heterocycle; an alkoxyoptionally substituted with an amino group, wherein the amino group isoptionally substituted with one or two C_(i) to C₆ alkyls, where thealkyls are optionally substituted with an amino optionally substitutedwith one or two C₁ to C₆ alkyls; an alkoxy optionally substituted with a5 to 8 membered heterocycle optionally substituted with a C₁ to C₆alkyl, which is optionally substituted with: an alkoxy; or an amino,optionally substituted with one or two C₁ to C₆ alkyls; or R₁ togetherwith R₂ forms:

or R₁ together with R forms:

and R₂ is: a methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, tert-butyl, n-pentyl, n-hexyl or cyclohexyl; a 5- or6-membered heterocycle; an amino optionally substituted with a C₁ to C₆alkyl; or R₁ together with R₂ forms:

or a pharmaceutically acceptable salt thereof
 36. The compound of claim35, wherein X is an amino group.
 37. The compound of claim 35, wherein Yis an amino group; a —SO₂R_(x), where R_(x) is methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexylor cyclohexyl; a —COOR_(x) group, where R_(x) is n-butyl, isobutyl,sec-butyl, tert-butyl, n-pentyl, n-hexyl, or cyclohexyl; a —COR_(a)group, where R_(a) is: an amino substituted with one or two C₁ to C₆alkyls, where the alkyls are optionally substituted with a C₆ aryl; a—NHR_(b) group where R_(b) is—a C₆ aryl optionally substituted with ahaloalkyl, a 5- or 6-membered heterocycle optionally substituted with aC₁ to C₆ alkyl; a C₁ to C₆ alkyl; or a —SR_(x) group, where R_(x) ismethyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,tert-butyl, n-pentyl, n-hexyl or cyclohexyl; a 5 or 6 memberedheteroaryl substituted with—a C₆ aryl; or a —NHCOR_(e) group where R_(e)is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,tert-butyl, n-pentyl, n-hexyl, cyclopentyl or cyclohexyl.
 38. Thecompound of claim 35, wherein R, R₁ and R₂ are independently methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl,n-pentyl, n-hexyl or cyclohexyl.
 39. The compound of claim 35, whereinsaid C₁ to C₆ alkyl in R, R₁ and R₂ is independently a methyl or anethyl.
 40. The compound of claim 39, wherein R₁ is selected from thegroup consisting of a methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl or cyclohexyl; and analkoxy optionally substituted with an amino group, wherein the aminogroup is optionally substituted with one or two C₁ to C₆ alkyls, wherethe alkyls are optionally substituted with an amino optionallysubstituted with one or two C₁ to C₆ alkyls; and an alkoxy optionallysubstituted with a 5 to 8 membered heterocycle optionally substitutedwith a C₁ to C₆ alkyl, which is optionally substituted with: an alkoxy;or an amino, optionally substituted with one or two C₁ to C₆ alkyls. 41.The compound of claim 35, wherein said compound is selected from thegroup consisting of:

or a pharmaceutically acceptable salt thereof
 42. A pharmaceuticalcomposition comprising a compound of claim 35, or a pharmaceuticallyacceptable salt thereof, together with a pharmaceutically acceptableexcipient.
 43. A pharmaceutical composition comprising a compound ofclaim 37, or a pharmaceutically acceptable salt thereof, together with apharmaceutically acceptable excipient.
 44. A pharmaceutical compositioncomprising a compound of claim 41, or a pharmaceutically acceptable saltthereof, together with a pharmaceutically acceptable excipient.
 45. Amethod of preventing or treating Hepatitis C viral infection comprisingadministering a compound of claim 35, or a pharmaceutical compositioncomprising a compound of claim
 42. 46. A method of preventing ortreating Hepatitis C viral infection comprising administering a compoundof claim 37, or a pharmaceutical composition comprising a compound ofclaim
 43. 47. A method of preventing or treating Hepatitis C viralinfection comprising administering a compound of claim 41, or apharmaceutical composition comprising a compound of claim 41.